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PubMed1991

Detection of Norwalk-like virus and specific antibody by immune-electron microscopy with colloidal gold immune complexes.

Direct electron-microscopy (DEM), immune electron microscopy (IEM) and four different procedures of immune electron microscopy with colloidal gold immune complexes were evaluated for the detection of Norwalk-like virus and specific antibody. A solid-phase immune electron microscopy with colloidal gold immune complexes-triple layer method (SPIEMGIC-TLM) is developed for screening patients' specimens for the detection of Norwalk-like virus and its specific antibody. The method demonstrates low non-specific background labelling and is simple, sensitive and easy to perform. A quadruple layer method (SPIEMGIC-QLM), which is a modification of the triple layer method, has been established by adding a cross-linking anti-IgG layer to amplify the reaction and to provide a more sensitive test which is suitable for screening monoclonal antibodies prepared against 32-34-nm Norwalk-like virus isolated in our laboratory.

Antibodies, Monoclonal↗
PubMed2005

Co-immunization of BALB/c mice with recombinant immunogens containing G protein fragment and chimeric CTL epitope of respiratory syncytial virus induces enhanced cellular immunity and high level of antibody response.

With the goal to develop effective immunogens against infection of respiratory syncytial virus (RSV), vectors co-expressing chimeric CTL epitope or G protein fragment of RSV with carrier protein DsbA (disulfide bond isomerase) were constructed. The capacity of the expressed recombinant immunogens to induce cellular and humoral immunities were evaluated. It was demonstrated that the presence of G protein fragment was able to enhance the CTL activities induced by the chimeric CTL epitope, though G protein fragment alone had no effect on induction of CTL response. In contrast, the level of antibody response to RSV and neutralization titer in co-immunization with G protein fragment plus chimeric CTL epitope was lower than that in immunization with G protein fragment alone. The challenge experiments indicated that co-immunization further reduced RSV titers both in lung tissue and nasal track, indicating the combination of humoral and cellular immunities is more effective. This data imply that the combination of the two protein immunogens would be a viable strategy for a RSV vaccine.

Animals↗
PubMed2005

Immunization with a gene encoding granulocyte-macrophage colony-stimulating factor inserted with a single helper T-cell epitope of an intracellular bacterium induces a specific T-cell subset and protective immunity.

We evaluated here the effect of immunization with a gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted with a helper T cell (Th) epitope, listeriolysin O (LLO) 215-226 derived from Listeria monocytogenes on induction of a specific Th by gene gun bombardment. Immunization of C3H/He mice with pGM215m plasmid encoding murine GM-CSF inserted with LLO 215-226 Th epitope gave the epitope-specific proliferative responses of CD4(+) T lymphocytes. In addition, specific interferon-gamma production from the splenocytes was observed. Concomitantly, pGM215m-immunized mice showed significant protective immunity against lethal listerial challenge. These results suggest that immunization of a gene for GM-CSF inserted with a Th epitope is useful for eliciting a specific Th subset in vivo.

Animals↗
PubMed2004

Immune responses of swine following DNA immunization with plasmids encoding porcine reproductive and respiratory syndrome virus ORFs 5 and 7, and porcine IL-2 and IFNgamma.

In the present study, five eukaryotic double-gene expression plasmids containing porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 and ORF7 genes combined with cDNAs encoding porcine IFNgamma and IL-2 were constructed for evaluation as PRRSV vaccine candidates. After immunization and viral challenge, two of three pigs immunized with pIRESorf5/IFNgamma, one of three pigs immunized with pIRESorf5/IL-2 and one of three pigs immunized with pIRESorf7/IL-2 were protected from lung lesions that were present in other vaccinated and control animals. Virus replication was reduced but not completely prevented in organs of the DNA-vaccinated animals as compared to controls. Therefore, the porcine cytokines IFNgamma and IL-2, delivered in combination with ORF5 or ORF7, may improve the immune efficacy of DNA vaccines against PRRSV.

Animals↗
PubMed2005

Toward a detailed characterization of feline immunodeficiency virus-specific T cell immune responses and mediated immune disorders.

Infection of domestic cats with feline immunodeficiency virus (FIV) is associated with the development of an acquired immunodeficiency syndrome (AIDS). The pathogenesis of FIV is not fully understood but it has been reported that the immune system is progressively impaired during disease progression. As a result, anti-FIV specific immune response will usually not clear the virus and the acute stage is followed by a chronic asymptomatic phase. The overall objective of this study was to characterized FIV-induced immune cellular responses and -mediated immune disorder following the first weeks post-infection. Using both cytokine ELISpot and intracellular staining assays, FIV-specific T cells were monitored at 6, 9 and 12 weeks post-infection. We demonstrated that both IFNgamma(+) and, CD4 and CD8 TNFalpha(+) T cells specifically respond to FIV antigens. These responses were found to reach a peak at 9 weeks post-infection. It was further shown that the TNFalpha(+)CD8(+) responding T cells were contained within a CD8beta(low)CD62L(-) T cell subpopulation, expanded in FIV-infected cats. This T cell subpopulation which present features of activated CD8 T cells was further shown to be susceptible to spontaneous apoptosis following a short-term in vitro culture. Moreover, it was observed that cell death by apoptosis of this T cell subset was increased following FIV antigen-recognition. Therefore, FIV might alter immune homeostasis in inducing chronic activation of TNFalpha(+)CD8(+) T cells which eventually will die following antigen contact while deleting CD4(+) T cells. Interestingly, this study confirmed the strong similarity between FIV and HIV pathogenesis.

Animals↗
PubMed2006

Cytokine responses to EHV-1 infection in immune and non-immune ponies.

Protecting equids against equine herpesvirus-1 (EHV-1) infection remains an elusive goal. Repeated infection with EHV-1 leads to protective immunity against clinical respiratory disease, and a study was conducted to measure the regulatory cytokine response (IFN-gamma and IL-4) in repeatedly infected immune ponies compared to non-immune ponies. Two groups of four ponies were established. Group 1 ponies had previously been infected on two occasions, and most recently 7 months before this study. Group 2 ponies had no history no vaccination or challenge infection prior to this study. Both groups were subjected to an intranasal challenge infection with EHV-1, and blood samples were collected pre-infection, and at 7 and 21 days post-infection for preparation of PBMCs. At each time point, the in vitro responses of PBMCs to stimulation with EHV-1 were measured, including IFN-gamma and IL-4 mRNA production, and lymphoproliferation. Group 1 ponies showed no signs of clinical disease or viral shedding after challenge infection. Group 2 ponies experienced a biphasic pyrexia, mucopurulent nasal discharge, and nasal shedding of virus after infection. Group 1 ponies had an immune response characterized both before and subsequent to challenge infection by an IFN-gamma response to EHV-1 in the absence of an IL-4 response, and demonstrated increased EHV-1-specific lymphoproliferation post-infection. Group 2 ponies had limited cytokine or lymphoproliferative responses to EHV-1 pre-challenge, and demonstrated increases in both IFN-gamma and IL-4 responses post-challenge, but without any lymphoproliferative response. Protective immunity to EHV-1 infection was therefore characterized by a polarized IFN-gamma dependent immunoregulatory cytokine response.

Animals↗
PubMed1994

Immunity to Haemophilus influenzae type b polysaccharide capsule in children with human immunodeficiency virus infection immunized with a single dose of Haemophilus vaccine.

We studied immunity to Haemophilus influenzae type b (Hib) polysaccharide capsule in 19 children infected with human immunodeficiency virus (HIV) immunized with a single dose of a Hib vaccine at a mean age of 28 months (range, 15 to 56 months). Four to eighty-five months after immunization, only 7 children (37%) were immune. There were no significant differences in the HIV classification of the Centers for Disease Control and Prevention, type of conjugate vaccine, age at vaccination or serologic testing, time from vaccination to antibody determination, and CD4 cell counts between children with and those without immunity.

Antibodies, Bacterial↗
PubMed2001

Immune response induced by immunization with Hepatitis B virus core DNA isolated from chronic active hepatitis patients.

There are many mutations in the gene encoding Hepatitis B virus (HBV) core antigen of chronic active hepatitis patients, and such mutations are most likely to be related to the severity of disease. Here, we constructed plasmids containing wild-type and deletion type of HBV core gene (HBc) to develop an experimental DNA vaccine and to compare immunogenicity of two types of HBc vaccine. Twenty-nine wild-types and seven deletion types of HBc were detected in sera of 32 Korean patients with chronic active hepatitis. Four wild-types (W1, W2, W4, W6) and two deletion types (D3, D4) of HBc were cloned into the pcDNA3 vector. Intramuscular immunization with wild-type HBc efficiently increased serum anti-HBc antibody response in a dose-dependent manner. Anti-HBc antibody response in mice injected with W6 increased 14 days after immunization, and peaked after 30 days and was maintained at least up to 50 days. W6 immunization induced a specific cytotoxic T lymphocyte response to W6-transfected 3LL (3LL-W6), and reduced the sizes of tumor mass of mice challenged with 3LL-W6 or 3LL transfected with D4. However, intramuscular immunization with D3 and D4 did not show antibody response at all. D3 and D4 have 157 bp (from 331 to 491 bp) and 122 bp (from 327 to 448 bp) gene deletion, respectively, and these encode class II MHC-restricted T-cell epitope. Altogether, these results suggest that mutant virus that has deleted HBc gene may evade immune systems due to loss of T-cell epitope.

Animals↗
PubMedDate not supplied

Manipulation of HIV-1 gp120-specific immune responses elicited via gene gun-based DNA immunization.

Gene gun-based DNA immunization using vectors encoding HIV-1 gp120 or influenza virus nucleoprotein result in Th2-like immune responses following successive immunizations. The codelivery of vectors encoding IL-2, IL-7, or IL-12 blocked this effect by markedly enhancing gp120-specific interferon gamma production, and suppressing IL-4 and IgG1 responses. An unbiased augmentation of all immune responses was observed by increasing the resting period between immunizations. In this case, IFN-gamma production following in vitro stimulation increased by over 1000-fold, while IL-4, IgG1, and IgG2a responses were elevated as well. Interestingly, cytokine gene codelivery, in the context of the longer resting period, provided no additional stimulation of Th1-like responses such as IFN-gamma and IgG2a production, although there was still some suppression of IL-4 production. These data demonstrate that the quality and magnitude of responses elicited following epidermal administration of DNA vaccines can be manipulated by multiple means.

AIDS Vaccines↗
PubMedDate not supplied

Induction of mucosal and systemic immune responses by intranasal immunization using recombinant cholera toxin B subunit as an adjuvant.

Intranasal (i.n.) immunization with Streptococcus mutans surface protein AgI/II mixed with cholera toxin B subunit (CTB) containing a trace amount of cholera toxin (CT) induces strong immune responses in mucosal and systemic sites, but whether pure CTB alone has an adjuvant effect has been questioned. To determine the adjuvant effect of recombinant (r) CTB, mice were immunized with 10 micrograms of AgI/II either mixed with or conjugated to 5 micrograms of rCTB, and antibody responses in saliva, nasal wash, gut wash, vaginal wash, and serum were assayed by ELISA. The results showed that AgI/II either mixed with or conjugated to rCTB could induce both mucosal IgA and systemic IgG antibodies to higher levels than in mice similarly immunized with AgI/II alone. Some responses, especially serum IgG antibodies, were enhanced by adding 5 micrograms CT to the immunogen, whereas overall mice immunized with AgI/II mixed with CTB contaminated with CT tended to generate the strongest mucosal IgA and serum IgG responses to AgI/II. However, rCTB used as an adjuvant induced lower antibody responses against itself than CTB intentionally or inadvertently mixed with CT. These results show that rCTB can serve as an adjuvant for protein immunogens administered by the i.n. route.

Adjuvants, Immunologic↗
PubMed1999

Immunity induced by DNA immunization with herpes simplex virus type 2 glycoproteins B and C.

The complete sequence of herpes simplex virus type 2 (HSV-2) glycoproteins B and C (gB & gC) were cloned into plasmid expression vectors and evaluated in murine and guinea pig genital HSV-2 models. Balb/c mice were immunized with either pgB-2 or pgC-2 plasmids intramuscularly (IM) or intradermally (ID). The vaccines induced HSV-2-specific neutralizing and ELISA IgG antibody, but little or no enhancement of viral clearance from the vagina was detected following intravaginal challenge. Immunization of guinea pigs with pgB-2 or pgC-2 induced ELISA IgG antibody; however, antibody titers were approximately one log(10) unit lower than that seen in HSV-2 convalescent sera. IM immunization of guinea pigs with either plasmid also did not decrease vaginal viral shedding following vaginal challenge, but the severity of the acute disease and the subsequent number of recurrent lesion days were reduced in animals immunized with pgB-2. Lastly, IM immunization of latently infected guinea pigs with a combined gB-2 and gC-2 plasmid vaccine significantly reduced the number of subsequent HSV-2 recurrences. DNA vectors expressing gB-2 or gC-2 were both immunogenic, although the gB-2 plasmid induced higher titers of antibody and significantly reduced primary and recurrent herpetic disease in the guinea pig model. These results also suggest that immunotherapy with plasmid expression vectors may be effective against recurrent genital HSV-2 disease.

Animals↗
PubMed2001

Fc gamma RIII mediates neutrophil recruitment to immune complexes. a mechanism for neutrophil accumulation in immune-mediated inflammation.

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fc gamma receptor IIIB (Fc gamma RIIIB), and the beta(2) integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of Fc gamma RIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, Fc gamma RIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with Fc gamma RIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Animals↗
PubMed2002

Genetics of the immune response: identifying immune variation within the MHC and throughout the genome.

With the advent of modern genomic sequencing technology the ability to obtain new sequence data and to acquire allelic polymorphism data from a broad range of samples has become routine. In this regard, our investigations have started with the most polymorphic of genetic regions fundamental to the immune response in the major histocompatibility complex (MHC). Starting with the completed human MHC genomic sequence, we have developed a resource of methods and information that provide ready access to a large portion of human and nonhuman primate MHCs. This resource consists of a set of primer pairs or amplicons that can be used to isolate about 15% of the 4.0 Mb MHC. Essentially similar studies are now being carried out on a set of immune response loci to broaden the usefulness of the data and tools developed. A panel of 100 genes involved in the immune response have been targeted for single nucleotide polymorphism (SNP) discovery efforts that will analyze 120 Mb of sequence data for the presence of immune-related SNPs. The SNP data provided from the MHC and from the immune response panel has been adapted for use in studies of evolution, MHC disease associations, and clinical transplantation.

Animals↗
PubMed1997

Immunity to Plasmodium falciparum malaria sporozoites by somatic transgene immunization.

Immunity against the human malaria parasite Plasmodium falciparum was induced using somatic transgene immunization, a method to effectively target B lymphocytes in vivo. A single inoculation of plasmid DNA containing an immunoglobulin heavy-chain gene coding in the complementarity-determining region 3 for three repeats of the sequence Asn-Ala-Asn-Pro (NANP), a B-cell epitope of P.falciparum sporozoites, induced antibodies against NANP in all mice. A booster with an antibody antigenized with the NANP peptide, or challenge with P. falciparum sporozoites, demonstrated the establishment of immunologic memory. Immunity to a parasite antigen can be induced by exploiting mechanisms in which B lymphocytes are both the source of the immunogen as well as the effector mechanism of immunity. The results indicate that somatic transgene immunization is a potential approach for vaccination against foreign pathogens.

Animals↗
PubMed1996

Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.

Prevention of sexually transmitted HIV infection was investigated in macaques by immunization with a recombinant SIV (simian immunodeficiency virus) envelope gp 120 and core p27 vaccine. In two independent series of experiments, we used the novel targeted iliac lymph node (TILN) route of immunization, aiming close to the iliac lymph nodes draining the genitorectal mucosa. Rectal challenge with the SIVmac 32H J5 molecular clone in two series induced total protection in four out of seven macaques immunized by TILN, compared with infection in 13 of 14 unimmunized macaques or immunized by other routes (P = 0.025). The remaining three macaques showed either a decrease in viral load ( > 90%) or transient viremia, indicating that all seven TILN-immunized macaques showed total or partial protection (P = 0.001). Protection was associated with significant increase in the iliac lymph nodes of IgA antibody-secreting cells to p27 (P < 0.02), CD8-suppressor factor (P < 0.01), and the chemokines RANTES and MIP-1 beta (P < 0.01).

Animals↗
PubMed2003

Hidden from the host: Spiroplasma bacteria infecting Drosophila do not cause an immune response, but are suppressed by ectopic immune activation.

Insects and other arthropods have an effective innate immune system that can clear infections with bacteria and other microorganisms. Despite this ability, one group of bacteria, the spiroplasmas, survive unharmed within the haemolymph of a wide range of arthropod hosts. We investigated the interaction between one member of this clade, a relative of Spiroplasma poulsonii, and the immune system of its Drosophila host. Expression of antimicrobial genes in spiroplasma-infected flies did not differ from wild-type controls either in the naturally infected state, nor after septic shock. We therefore concluded that spiroplasma infection did not induce an immune response in its host, but that this absence of response was unlikely to be because the bacterium inhibited response. Further experiments revealed immune reactions induced ectopically did reduce parasite titre. We therefore conclude that this bacterium has a novel form of interaction with its host, being hidden from the host immune system, but potentially suppressible by it.

Animals↗
PubMed2002

Immune responsiveness in renal transplant recipients: mycophenolic acid severely depresses humoral immunity in vivo.

BACKGROUND: Current immunosuppressive drug treatments for renal transplant recipients result in high one-year graft survival rates. Despite adequate suppression of the immune response directed to the allograft, the immune system remains able to cope with many infectious agents. METHODS: To define the influence of distinct immunosuppressive treatment protocols, primary and secondary cellular and humoral immune responses in groups of renal transplant recipients were studied: patient treated with prednisolone and cyclosporine A (P/CsA); with IgA CD3 monoclonal antibody as a rejection treatment superimposed on prednisolone and cyclosporine A (IgA CD3 mAb+P/CsA); and with prednisolone, cyclosporine A and mycophenolate mofetil (P/CsA/MMF). RESULTS: Primary in vitro proliferative responses to the protein antigen keyhole limpet hemocyanin (KLH) were not significantly disturbed in P/CsA treated patients, or in IgA CD3 mAb+P/CsA and P/CsA/MMF treated patients. In vitro proliferative responses to the recall antigen tetanus toxoid (TT) were similarly unaffected. Antigen-specific antibody responses to immunization with KLH and TT were not affected by treatment with P/CsA, or by IgA CD3 mAb+P/CsA, but were severely disturbed in patients treated with P/CsA/MMF. All patients displayed a profound inhibition of the delayed-type hypersensitivity skin reactivity to KLH and recall antigens. Nevertheless, in most patients with P/CsA treatment, T cell infiltrates were observed in skin biopsies from the site of KLH challenge, while expression of intercellular cell adhesion molecule-1 (ICAM-1) expression in challenged skin was significantly decreased in these patients. The balance between T helper 1 and T helper 2 cells was unaffected by immunosuppressive treatments during one year of follow-up. CONCLUSIONS: Immunosuppressive drug treatment with P/CsA inhibits delayed-type hypersensitivity skin reactions to both primary and frequently encountered antigens. Histological studies indicate an effect on ICAM-1 expression, leaving the influx of CD3pos T cells unaffected. Administration of a 10-day course of IgA CD3 mAb does not add profound immunosuppressive effects on the measured parameters. In contrast, addition of treatment with MMF profoundly decreases both primary and secondary humoral immune responsiveness in vivo. Finally, no effect of the studied immunosuppressive drugs on Th1/Th2 balance in vivo was measured.

Adolescent↗
PubMed2001

Increased levels of immune inhibitory CD34+ progenitor cells in the peripheral blood of patients with node positive head and neck squamous cell carcinomas and the ability of these CD34+ cells to differentiate into immune stimulatory dendritic cells.

OBJECTIVES: This study determined whether mobilization of immune inhibitory CD34+ cells by head and neck squamous cell carcinomas (HNSCC) is most prominent in patients who are node positive and whether these CD34+ cells could differentiate into immune stimulatory dendritic cells. STUDY DESIGN AND SETTING: Peripheral blood from patients with head and neck cancer was used to measure the frequency of CD34+ cells and their capacity to differentiate into immune stimulatory dendritic cells. RESULTS: This study demonstrated that increased CD34+ cell levels were most prominent in patients who were node positive and patients with recurrent disease. These CD34+ cells differentiated into dendritic cells that were able to present tetanus toxoid to autologous T-cells. CONCLUSIONS: Immune suppressive CD34+ cells that are prominent in patients with HNSCC who are node positive are able to develop into immune stimulatory dendritic cells. SIGNIFICANCE: Differentiation of tumor-mobilized CD34+ cells into dendritic cells may be an immunotherapeutic approach to stimulate antitumor reactivity.

Antigen Presentation↗