Effect of pregnancy plasma upon in vitro parameters of cell mediated immunity

Pregnant women were classified according to their serological status for cytomegalovirus, herpes simplex virus or rubella virus. Lymphocytes taken from non-pregnant women were shown to be able to recognise viral antigens and the mitogen phytohaemagglutinin by the measurement of proliferative responses and by the production of gamma interferon. Proliferative responses or gamma interferon production were greatly reduced in the presence of plasma taken during the first, second or third trimester and immediately post-partum. The responses then gradually returned to normal after delivery. The availability of serial sera taken before pregnancy as well as during and after pregnancy in individual women showed that this effect was maintained even when sera had been stored frozen for more than one year. Mixing experiments were performed to vary the proportion of pregnancy serum in any particular assay but this did not prove that pregnancy sera were actively suppressive. Instead, the data suggest that pregnancy sera are deficient in some factor or factors which are required to support lymphocyte proliferation. The effect was not attributable to the physiological haemodilution of pregnancy leading to a reduced concentration of putative factors nor could transferrin levels or the iron binding capacity of this protein be implicated.     

Transplacental transmission of BK virus in human.

The prevalance and distribution of BK virus antibody in women during pregnancy and the occurrence of transplacental transmission of BK virus was determined by measurement of IgM antibody in the serum. Sera were collected from 63 nonpregnant women, 71 women who had experienced spontaneous abortion, 80 in the first trimester of pregnancy and the same 80 at delivery. Umbilical cord blood was also taken at delivery. Hemagglutination inhibition (HI) tests for BK virus used the micromethod of Gardner. Results indicate that a significant level of HI antibody was present in 70% of sera from all 4 experimental groups. This showed that BK virus infection was not limited to cases of spontaneous abortion. Of the 80 pregnant mothers, 6 showed a 4-fold or greater HI antibody seroconversion to BK virus after delivery. Of these 6 seroconversion patients, sensitive antibody was detected in 3 umbilical cords. Umbilical cords of those without seroconversion had no sensitive antibody. As evidenced by 2-NE-sensitive antibody, BK virus infections were also recognized in 6 of 71 women who aborted, 4 of 80 in the first trimester of pregnancy and 2 collected after delivery. The 2-ME-sensitive antibody was not found in any of 63 samples from nonpregnant women. Data indicate that 2-ME-sensitive antibody was present only in sera of women during pregnancy and after abortion. It may be possible that BK virus persists in a latent form in many healthy women and becomes activated during pregnancy.     

Transient mobilization of human immunodeficiency virus (HIV)-specific CD4 T-helper cells fails to control virus rebounds during intermittent antiretroviral therapy in chronic HIV type 1 infection

Immune control of human immunodeficiency virus (HIV) is not restored by highly active antiretroviral therapies (HAART) during chronic infection. We examined the capacity of repeated structured therapeutic interruptions (STI) to restore HIV-specific CD4 and CD8 T-cell responses that controlled virus production. Eleven STI (median duration, 7 days; ranges, 4 to 24 days) were performed in three chronically HIV-infected patients with CD4 counts above 400/mm(3) and less than 200 HIV RNA copies/ml after 18 to 21 months of HAART; treatment resumed after 1 week or when virus became detectable. HIV-specific T-cell responses were analyzed by proliferation, gamma interferon (IFN-gamma) production, and enzyme-linked immunospot assays. Seven virus rebounds were observed (median, 4,712 HIV-1 RNA copies/ml) with a median of 7 days during which CD4 and CD8 counts did not significantly change. After treatment resumed, the viral load returned below 200 copies/ml within 3 weeks. Significant CD4 T-cell proliferation and IFN-gamma production against HIV p24 appeared simultaneously with or even before the virus rebounds in all patients. These CD4 responses lasted for less than 3 weeks and disappeared before therapeutic control of the virus had occurred. Increases in the numbers of HIV-specific CD8 T cells were delayed compared to changes in HIV-specific CD4 T-cell responses. No delay or increase in virus doubling time was observed after repeated STI. Iterative reexposure to HIV during short STI in chronically infected patients only transiently mobilized HIV-specific CD4 T1-helper cells, which might be rapidly altered by virus replication. Such kinetics might explain the failure at delaying subsequent virus rebounds and raises concerns about strategies based on STI to restore durable HIV-specific T-cell responses in chronic HIV infection.     

Late pregnancy suppresses relapses in experimental autoimmune encephalomyelitis: evidence for a suppressive pregnancy-related serum factor

Women with multiple sclerosis have significantly diminished disease activity during pregnancy. The purpose of our study was to identify the underlying mechanism for the diminished disease activity. We found that during the period of late pregnancy there is protection against paralysis, during both the induction and effector phases of relapsing experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. We did not find any changes in the cytokine secretion profiles or the proliferative activity of autoreactive T cells from mice induced during late pregnancy compared with virgin controls. In mice mated after disease onset, the inflammatory histologic lesions did not clear, despite marked clinical improvement during pregnancy. We found evidence for a serum factor present in late pregnancy that suppresses T cell activation. In the presence of sera taken from mice late in pregnancy, the proliferative response and IL-2 production of proteolipid protein p139-151-specific T cells were significantly diminished as compared with stimulation in the presence of normal mouse sera. In conclusion, serum from late pregnancy has the capacity to down-regulate T cell responses and might be associated with the amelioration of disease activity in experimental autoimmune encephalomyelitis.     

An immunochemical demonstration of a pregnancy-specific protein in the horse and its use in the serological detection of early pregnancy

Two-dimensional crossed immunoelectrophoresis of sera from pregnant and non-pregnant horses, using antisera developed against early pregnant mare serum, revealed the presence of two immunologically related proteins one of which appeared to be specific to the pregnant state. This pregnancy-specific protein had beta 2-electrophoretic mobility and was first detectable at Day 6 after successful mating with a stallion. The second protein had gamma 2-electrophoretic mobility and was present in sera from pregnant and non-pregnant horses. The proteins were termed beta 2-horse pregnancy protein and gamma 2-horse protein respectively. The latter appeared to be immunologically related to the former in that the precipitin lines of the 2 proteins showed continuity. Samples from 16 mares mated with a stallion were investigated for the beta 2-protein during the first 3 weeks after mating. Of the 11 successful matings, confirmed by ultrasonic scanning at 90 days or by a successful outcome, 10 mares showed the presence of the protein. In all of 14 non-pregnant sera taken from mares not recently mated, the protein was not detectable. The validity of detection of beta 2-protein as an indication of pregnancy was clinically significant at the 10% level. The presence of the protein in 2 out of the 5 recently mated mares that did not become pregnant may be indicative of a biochemical pregnancy that failed at a later stage of gestation.     

Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production.

Earlier studies from this laboratory showed that infection of BALB/cByJ mice by a natural route with mouse hepatitis virus, strain JHM (MHV-JHM), results in functional splenic T cell suppression in vitro. This was evidenced by reduced concanavalin A-driven spleen cell proliferation and interleukin (IL)2 production measured after conventional intervals of cell culture (72 and 24 h, respectively). The purpose of the present work was to determine whether MHV-induced T cell dysfunction is kinetic or absolute and whether production of other T-cell derived cytokines is defective. Bioassays revealed that production of IL2, gamma interferon, and IL4 by spleen cells from acutely infected mice is suppressed and that some of the defects are kinetic as well as absolute. Proliferative responses of both CD4+ and CD8+ T cells were depressed, but neither cell type contained infectious virus. Cells that proliferated poorly in response to concanavalin A were fully capable of responding to specific virus stimulation. These results further emphasize the potential complications that MHV infection may pose to immunologic research using mice.     

Amplification of rabies virus-induced stimulation of human T-cell lines and clones by antigen-specific antibodies.

The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. Experiments performed on T-cell clones specific for either rabies glycoprotein or nucleocapsid showed that immune plasma as well as antiglycoprotein and antinucleoprotein murine monoclonal antibodies possessed the capacity to increase significantly the antigen-induced proliferative responses of these clones. The overall results indicate that this in vitro effect of antigen-specific antibodies on the response of regulatory T lymphocytes to rabies virus could be an important factor in the development of effective immune responses in vivo to rabies virus.     

Synthesis of type C virus particles from murine-cultured cells induced by iododeoxyuridine. V. Effect of interferon and its interaction with dexamethasone.

Previous studies have shown that in certain cell systems dexamethasone may enhance the production of type C viruses. Conversely, interferon has been shown to inhibit their production. Both appear to exert their influence late in the viral replication cycle rather than on the synthesis of viral-specific RNA. In this report dexamethasone and interferon have been used to study some aspects of the mechanisms involved in the synthesis of type C viruses in murine K-BALB cells following induction of virus production by iododeoxyuridine. Interferon inhibited production of xenotropic type C virus induced by iododeoxyuridine from K-BALB cells both in the absence and presence of dexamethasone, but it did not affect production of N-tropic type C virus. Exposure of the cells to interferon for longer than 12 h was required for maximum effect. Two types of inhibitory effects were observed: one diminished by dexamethasone when the steroid was added 24 h after interferon removal, and the second resistant to dexamethasone. The concentration of intracellular group-specific antigen was diminshed after interferon and increased after dexamethasone exposure. When induced cells were treated with both interferon and dexamethasone, the intracellular group-specific protein concentration was slightly increased, but virus production was reduced 10-fold compared with induced cells treated with dexamethasone alone. We conclude that interferon and dexamethasone may affect both the synthesis of viral proteins and the assembly or release of virus particles and that dexamethasone can partially nullify the inhibitory activity of interferon. The results also support previous conclusions that the regulatory mechanisms for synthesis of viral proteins and for the release of viral particles may differ and that controls for xenotropic and ecotropic virus formation may not be identical.     

Interferon production in chick-embryo cells. The effect of puromycin and p-fluorophenylalanine

1. When chick-embryo cells were treated with ultraviolet-inactivated influenza virus (Melbourne strain), interferon was produced after a lag period of about 10hr. 2. The addition of small amounts of either puromycin or p-fluorophenylalanine immediately after the virus inhibited the subsequent production of interferon. Both inhibitors primarily affected protein synthesis, and it is concluded that interferon production involves new protein synthesis. 3. Results obtained by the addition of either inhibitor for short periods during the lag phase demonstrated a requirement for protein synthesis during the second half of the lag phase. 4. Addition of puromycin during the course of interferon production caused almost immediate inhibition, but interferon formation became insusceptible to the action of p-fluorophenylalanine at about 26hr. after infection. Possible explanations of this effect are discussed.     

Antibacterial activity of BCG-induced, interferon-containing sera

Sera from mice which have been vaccinated with BCG and challenged with old tuberculin contain gamma interferon. These same sera also express antibacterial activity. Using Staphylococcus aureus we demonstrated that its growth was inhibited at dilutions of sera as high as 1:320. A 4% concentration of sera reduced the growth rate of the S. aureus from 1.6 to 0.6 doubling times per hour. The activity was stable at 56 degrees C but destroyed by 80 degrees C. It was nondialysable and destroyed by acid conditions (pH 2.0) and by the proteolytic enzymes trypsin and chymotrypsin. Antibodies to gamma interferon neutralized the antiviral activity but not the antibacterial activity. Mitogen-induced and virus-induced interferons did not have activity. We subsequently demonstrated that the factor could be induced in mice using BCG without the secondary old tuberculin challenge. No gamma interferon was found in the sera of mice given BCG without old tuberculin. These findings indicate that the antibacterial activity of these sera is not dependent on the presence of gamma interferon. We will continue to work to characterize and identify the antibacterial component in these sera.     

Alpha/Beta and Gamma Interferons Are Induced by Infection with Noncytopathic Bovine Viral Diarrhea Virus In Vivo

In contrast to the results of previous in vitro studies, experimental infection of calves with noncytopathic bovine viral diarrhea virus (ncpBVDV) was found to induce strong alpha/beta and gamma interferon responses in gnotobiotic animals. These responses were associated with depressed levels of transforming growth factor beta (TGF-beta) in serum. The results of this study indicate that the immunosuppression caused by ncpBVDV is not associated with low interferon responses or elevated levels of TGF-beta.     

Effect of Statolon-induced Interferon on Antibody Formation

The effect of statolon-induced interferon on immune response was investigated. Young adult Swiss albino mice were immunized with chicken red blood cells or infectious mengovirus. The red blood cells were injected three times and the virus twice at 2-week intervals, with or without treatment with statolon 24 hr before the inoculations. The effect of interferon on antibody formation was measured by determinations of hemagglutinating, hemolytic, and virus-neutralizing antibodies in the sera of treated and nontreated groups. The hemagglutinin and hemolysin titers after each successive inoculation were equal in both groups, indicating that interferon had no effect on their production. A decrease in the primary response to the viral antigen was associated with interferon induction; the booster response was unaffected. The possible use of interferon inducers as attenuants for immunization with live virulent viruses is discussed.     

Importance of interferons in recovery from mousepox.

Gamma interferon is shown to be critical in recovery of C57BL/6 mice from mousepox. Anti-gamma interferon treatment of mice infected in the footpad with ectromelia virus resulted in enhanced spread to and efficient virus replication in the spleen, lungs, ovaries, and, especially, liver. All treated, infected mice died within a mean of 7 days, 2.5 days earlier than mice with severe combined immunodeficiency that were given a comparable infection. On the other hand, alpha interferon appeared not to have a major role in controlling virus replication in tissues examined, and beta interferon was important for virus clearance in the liver and ovaries but not the spleen. Either anti-alpha, beta interferon or anti-beta interferon antibody therapy resulted in only 25% mortality. Infected control mice survived but showed persistence of ectromelia virus at the site of infection (the footpad) and transient presence of the virus in the spleen, liver, lungs, and ovaries and in the fibroreticular but not lymphoid cells of the draining popliteal lymph node. Depletion of gamma interferon but not alpha and/or beta interferon resulted in a significant reduction in the numbers of splenic T (especially gamma delta-TCR+), B, and Mac-1+ cells, although the proportion of Mac-1+ cells in the spleen increased compared with control values. Depletion of alpha, beta, or gamma interferons did not severely affect the generation of virus-specific cytotoxic T-lymphocyte responses or natural killer cell cytolytic activity. This study, in which a natural virus disease model was used, underscores the crucial importance of gamma interferon in virus clearance at all stages of infection and in all tissues tested except the primary site of infection, where virus clearance appears to be delayed.     

Protective effect of measles virus inoculation on subacute sclerosing panencephalitis virus-infected mice

The Biken strain of subacute sclerosing panencephalitis (SSPE) virus caused a fatal neurologic disease in adult mice after intracerebral inoculation. However, the mice were completely protected from the disease when a high dose of measles virus was given intracerebrally after the SSPE virus infection. The measles virus inoculation induced interferon production and immune responses. An experiment with athymic nude mice showed that interferon and anti-measles antibody were able to prolong the incubation period of the disease but not to protect the SSPE virus-infected nude mice from death. For complete protection, T lymphocytes appeared to be essential. The present study suggested that the protective effect of measles virus inoculation is basically due to the induction of immune responses and that SSPE virus infection in mice is susceptible to immune reactions.     

Inhibition of human immunodeficiency virus type 1 gp120 presentation to CD4 T cells by antibodies specific for the CD4 binding domain of gp120

Human immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120(CD4BD)), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120(CD4BD) MAbs. The anti-gp120(CD4BD) MAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120(CD4BD) MAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120(CD4BD) antibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120(CD4BD) Abs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs.     

Impact of mucosal inflammation on cervical human immunodeficiency virus (HIV-1)-specific CD8 T-cell responses in the female genital tract during chronic HIV infection

The female genital tract is the major route of heterosexual human immunodeficiency virus (HIV) acquisition and transmission. Here, we investigated whether HIV-specific CD8 T-cell-mediated immune responses could be detected in the genital mucosa of chronically HIV-infected women and whether these were associated with either local mucosal HIV shedding or local immune factors. We found that CD8⁺ T-cell gamma interferon responses to Gag were detectable at the cervix of HIV-infected women but that the magnitude of genital responses did not correlate with those similarly detected in blood. This indicates that ex vivo HIV responses in one compartment may not be predictive of those in the other. We found that increased genital tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) levels correlated significantly with levels of Gag-specific CD8⁺ T cells at the cervix. Women who were detectably shedding virus in the genital tract had significantly increased cervical levels of TNF-α, IL-1β, IL-6, and IL-8 compared to women who were not detectably shedding virus. We were, however, unable to detect any association between the magnitude of cervical HIV-specific responses and mucosal HIV shedding. Our results support the hypothesis that proinflammatory cytokines in the female genital tract may promote HIV replication and shedding. In addition, we further show that inflammatory cytokines are associated with increased levels of HIV-specific CD8 effector cells at the genital mucosa but that these were not able to control genital HIV shedding.     

In vivo blockade of gamma interferon affects the influenza virus-induced humoral and the local cellular immune response in lung tissue.

Influenza virus infection induces the local production of gamma interferon (IFN-gamma) by T cells and non-T cells in the respiratory tract. To elucidate the possible functions of this cytokine, the humoral and local cellular immune responses to influenza virus were studied in BALB/c mice with or without in vivo neutralization of IFN-gamma by using monoclonal antibodies. Neutralization of IFN-gamma led to a significant reduction in virus-specific titers of immunoglobulins G2a and G3 in serum but had little effect on other isotypes. Studies on cells isolated from the lung parenchyma itself revealed that at the height of the immune response the ability of these cells to produce cytokines after antigen or T-cell receptor/CD3 stimulation was not affected. Ex vivo cytolytic activity by lung parenchyma cells, which is induced by infection with this virus in normal mice, was also found to be undisturbed by this treatment, even though anti-IFN-gamma antibody activity was recovered from lung lavage samples and sera at all days studied. Surprisingly, in vivo neutralization of IFN-gamma led to a significant reduction in the magnitude of the cellular infiltrate in the lung tissue which followed infection, suggesting an involvement of IFN-gamma in the mechanisms that regulate increased leucocyte traffic in the inflamed lung parenchyma. This conclusion was supported by findings of differences between mock-treated and anti-IFN-gamma-treated mice in the number of CD8+ lung T cells expressing CD49d (alpha4-integrin) and CD62L at various times after influenza virus infection. This study therefore demonstrates that IFN-gamma affects the local cellular response in the respiratory tract as well as the systemic humoral response to influenza virus infection.     

Interleukin-12 gene expression after viral infection in the mouse.

Interleukin-12 is a lymphokine that triggers gamma interferon secretion by various cells and differentiation of T-helper lymphocytes towards the Th1 subtype. Since viruses are potent inducers of gamma interferon production and elicit immune responses most probably mediated by Th1 cells, like B-cell immunoglobulin G2a secretion, we analyzed interleukin-12 message expression after infection of mice with lactate dehydrogenase-elevating virus, mouse hepatitis virus, and mouse adenovirus. Our results indicated that the message for the p40 component of interleukin-12 was transiently increased shortly after infection. Interleukin-12 was expressed mainly by macrophages. Therefore, production of interleukin-12 might constitute the initial event that would determine the subsequent characteristics of the immune response elicited by viral infections.     

Prevalence of antibody to human herpesvirus 6 in women and children

The antibody prevalence to human herpesvirus 6 (HHV-6) was compared between pregnant women and control women of similar ages in Thailand. No significant difference was detected in the antibody positive rate and antibody titers between both groups. The antibody titers in sera collected from pregnant women at 1st and 3rd trimester remained unchanged. Next, the antibody prevalence in infants were examined and the positive rate decreased until 3 months and started to increase from 6 months after birth. The present results suggest that the reactivation of HHV-6 might not occur during pregnancy and this virus infects infants postnatally.     

CpG Oligodeoxynucleotides with Hepatitis B Surface Antigen (HBsAg) for Vaccination in HBsAg-Transgenic Mice

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage. In control nontransgenic C57BL/6 mice, three immunizations with HBsAg and CpG triggered the production of anti-HBs antibodies and of HBs-specific T cells that secrete gamma interferon but do not display any HBsAg-specific cytotoxic activity. In the HBsAg-transgenic mice, immunization with HBsAg and CpG oligodeoxynucleotides, but not with CpG alone, induced the clearance of HBsAg circulating in the sera, with a concomitant appearance of specific antibodies, and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Finally, adoptive transfer experiments with CD8(+) T cells primed in C57BL/6 mice with HBsAg and CpG oligodeoxynucleotide-based immunization show that these cells were able to partially control transgene expression in the liver and to clear the HBsAg from the sera of recipient transgenic mice without an antibody requirement. CpG oligodeoxynucleotides motifs combined with HBsAg could therefore represent a potential therapeutic approach with which to treat chronically infected patients.