Protective role of IL-22 against Schistosoma mansoni soluble egg antigen-induced granuloma in Vitro.

The role of T helper-17 (Th17) lymphocytes in the regulation of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma is unknown. This study examined the effect of Th17 cytokines (IL-17 and IL-22) on granulocyte recruitment and functions during SEA-induced granuloma formation in vitro in Schistosoma-infected and noninfected individuals. Granulocytes were isolated from 27 Schistosoma-infected patients and 13 controls and were used for granuloma induction using SEA-conjugated polyacrylamide beads in the presence of Th17 cytokines. Granuloma index was assessed, and granulocyte mediators such as tumour necrosis factor (TNF-α), hydrogen peroxide (H2 O2 ) and nitric oxide (NO) were measured in the culture supernatant at the 7th day using enzyme-linked immunosorbent assay (ELISA). Schistosoma-infected patients had significant larger SEA-induced granuloma than controls. IL-17 (125 pg/mL) induced the optimum size for granuloma within 3-7 days. However, IL-22 at different concentrations up to 300 pg/mL had no effect on granuloma formation. Using both cytokines simultaneously, IL-22 suppressed the effect of IL-17 and prevented granuloma formation. IL-17 significantly decreased TNF-α, H2 O2 and NO levels in Schistosoma-infected individuals. In contrast, IL-22 increased TNF-α and H2 O2 levels. In conclusion, IL-17 accelerates SEA-induced granuloma formation and inhibits granulocytes functions in Schistosoma-infected patients, while IL-22 inhibited the granuloma formation, but enhanced granulocyte functions.

Hepatitis E infection in HIV-infected liver and kidney transplant candidates.

Hepatitis E virus (HEV) has been reported to cause acute and chronic hepatitis in those with HIV infection and among solid organ transplant recipients in Europe. Limited data indicate that HEV is endemic in the United States, but the prevalence and significance of HEV infection among those with HIV and awaiting solid organ transplantation is unknown. We evaluated anti-HEV IgM and IgG antibodies and HEV RNA in 166 HIV-infected solid organ transplant candidates enrolled in the NIH HIV-Transplant Cohort. Overall prevalence of anti-HEV IgG approached 20% in both liver and renal transplant candidates. Evidence of recent infection was present in approximately 2% of liver transplant candidates and none of the kidney transplant candidates. HEV RNA was not detected in any patient. We conclude that markers of HEV infection are frequent among candidates for transplantation, but active, ongoing viremia is not seen. Evidence of recent infection (acute on chronic) liver disease was present in liver but not kidney recipients.

Mutations associated with occult hepatitis B virus infection result in decreased surface antigen expression in vitro.

Occult hepatitis B virus (HBV) infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild-type HBsAg expression vectors were constructed from genotype-matched chronic HBV sequences. Site-directed mutagenesis was then utilized to introduce three genotype A mutations - M103I, K122R and G145A - associated with occult HBV infection in vivo, alone and in combination, into the wild-type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all three mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.

Genomic variability associated with the presence of occult hepatitis B virus in HIV co-infected individuals.

Occult hepatitis B virus (O-HBV) infection is characterized by the presence of HBV DNA without detectable hepatitis B surface antigen (HBV DNA+/HBsAg-) in the serum. Although O-HBV is more prevalent during HBV/HIV co-infection, analysis of HBV mutations in co-infected patients is limited. In this preliminary study, HBV PreSurface (PreS) and surface (S) regions were amplified from 33 HIV-positive patient serum samples - 27 chronic HBV (C-HBV) and six O-HBV infections. HBV genotype was determined by phylogenetic analysis, while quasispecies diversity was quantified for the PreS, S and overlapping polymerase regions. C-HBV infections harboured genotypes A, D and G, compared to A, E, G and one mixed A/G infection for O-HBV. Interestingly, nonsynonymous-synonymous mutation values indicated positive immune selection in three regions for O-HBV vs one for C-HBV. Sequence analysis further identified new O-HBV mutations, in addition to several previously reported mutations within the HBsAg antigenic determinant. Several of these O-HBV mutations likely contribute to the lack of detectable HBsAg in O-HBV infection by interfering with detection in serologic assays, altering antigen secretion and/or decreasing replicative fitness.

Effect of low-dose IL-2 immunotherapy on frequency and phenotype of regulatory T cells and NK cells in HIV/HCV-coinfected patients.

We evaluated the effect of low-dose IL-2 therapy (daily 1.2 MIU/m(2), subcutaneously) on the number and phenotype of regulatory T cells (T(regs)) and natural killer (NK) cells in HIV/HCV-coinfected patients taking antiretroviral therapy. The frequency and phenotype of circulating T(regs) (defined as CD3(+) CD4(+) CD25(high) or CD3(+) CD4(+) FOXP3(+)) and NK cells (CD3(-) CD16(+)/CD56(+)) were evaluated at baseline and after 12 weeks of treatment. The expression of CD25, CTLA-4, and granzymes A and B by CD4(+) FOXP3(+) cells, as well as the expression of KIR receptors (NKB1, CD158a, and NKAT2) on NK cells, was evaluated. Low doses of IL-2 resulted in the augmented frequency and absolute number of T(regs) in coinfected individuals. FOXP3 levels per cell as well as augmented CD25 and CTLA-4 expression by T(regs) suggested that IL-2 may lead to both expansion and activation of T(regs), although changes in the proportion of CD4(+) FOXP3(+) cells were not associated with changes in HCV viral load and CD4(+) cells between baseline and week 12. NK cell frequency also increased after IL-2 therapy. Interestingly, the pattern of expression of KIR receptors was changed by IL-2 treatment, since the frequency of NK cells expressing NKB1 augmented whereas the frequency of NK expressing CD158a and NKAT2 decreased.

Characterization of hepatitis E-specific cell-mediated immune response using IFN-gamma ELISPOT assay.

In developing countries, hepatitis E (HEV) and hepatitis A (HAV) are the major causes of acute viral hepatitis with similar feco-oral modes of transmission. In contrast to the high seroprevalence of hepatitis A infection, a low seroprevalence of HEV among children in endemic areas has been reported. These data suggest the possibility that silent HEV infection is undiagnosed by the current available methods. Many of the serological tests used for HEV diagnosis have poor specificity and are unable to differentiate among different genotypes of HEV. Moreover, the RT-PCR used for HEV isolation is only valid for a brief period during the acute stage of infection. Cell-mediated immune (CMI) responses are highly sensitive, and long lasting after sub-clinical infections as shown in HCV and HIV. Our objective was to develop a quantitative assay for cell-mediated immune (CMI) responses in HEV infection as a surrogate marker for HEV exposure in silent infection. Quantitative assessment of the CMI responses in HEV will also help us to evaluate the role of CMI in HEV morbidity. In this study, an HEV-specific interferon-gamma (IFN-gamma) ELISPOT assay was optimized to analyze HEV-specific CMI responses. We used peripheral blood mononuclear cells (PBMC) and sera from experimentally infected chimpanzees and from seroconverted and control human subjects to validate the assay. The HEV-specific IFN-gamma ELISPOT responses correlated strongly and significantly with anti-HEV ELISA positive/negative results (rho=0.73, p=0.02). Moreover, fine specificities of HEV-specific T cell responses could be identified using overlapping HEV ORF2 peptides.

Absence of high-affinity binding sites for interferon alpha/beta in variant murine CD4+ T lymphocytes not expressing the T cell antigen receptor.

The T cell antigen receptor complex (CD3/Ti) plays a role in specific antigen recognition as well as in signal transduction, with its surface expression required for the function of several other structurally distinct receptor systems, including CD2, Ly-6(TAP), and Thy-1. In this communication, evidence is presented suggesting an association between the surface expression of CD3/Ti and that of the type 1 interferon (IFN) receptor in a CD4+ murine T cell clone. We tested the proliferative responses and their capacity to be inhibited by type 1 IFN with the wild-type, CD3/Ti-positive T cell clone and its CD3/Ti-negative variants did not respond to specific antigen or anti-CD3 antibody stimulation but they did respond to T cell growth factor (TCGF), stimulation as did the wild-type parental cells. Therefore, the type 1 IFN inhibition of TCGF-stimulated proliferative responses of wild-type and variant cells were compared. Both natural and recombinant type 1 IFNs inhibited TCGF-induced tritiated thymidine (3H-TdR) incorporation in the wild-type T cell clone, with a ID50 of 60-80 U/ml. By contrast, the variants required much higher doses of type 1 IFN. The ID50 with natural murine IFN-beta was 10,000 U/ml, but this same dose of human IFN-alpha A/D gave only a marginal inhibitory effect. Accompanying the loss of IFN responsiveness, these variants also exhibited a loss of high-affinity type 1 IFN receptors. Taken together, these data suggest that the CD3/Ti complex plays a role in the surface expression of the type 1 IFN receptor in a CD4+ T cell clone.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoprophylaxis of hepatitis C virus infection.

Because hepatitis C virus is etiologically involved in about half the cases of the world's most common cancer, hepatocellular carcinoma, and because this virus is likely to continue to spread in most of the developing world for many years, the authors believe that development of a prophylactic vaccine is imperative. Numerous approaches are available to overcome the many impediments which make the development of an HCV vaccine difficult. Such impediments include the many viral genotypes and quasispecies of HCV and the association of virions with host lipids. It is likely that overcoming these impediments will require a vaccine which induces a strong cell-mediated response. The most promising approach seems to be DNA-based immunization or a prime-boost regimen with DNA priming and boosting with a viral vector. Potentiation of responses with adjuvant strategies will probably be necessary. Hepatitis C virus immunization is in an early stage of development. Given the explosive growth in the understanding of immunology, progress should be rapid.

Progress toward the development of a bacterial vaccine vector that induces high-titer long-lived broadly neutralizing antibodies against HIV-1.

Conformationally constrained HIV-1 Env and gp120 immunogens induce broadly cross-reactive neutralizing antibodies. Thus, it is now feasible to rationally design an HIV-1 vaccine that affords protection through humoral mechanisms. This paper reviews our progress toward the development of an oral bacterial vaccine vector that is capable of delivering an HIV-1 DNA vaccine to host lymphoid tissues and inducing broadly neutralizing antibodies to HIV-1 in the mucosal and systemic immune compartments.

Prevalence of anti-HEV IgM among blood donors in Egypt.

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in developing countries. In Egypt; where up to 80% of the inhabitants of rural villages have anti-HEV antibodies denoting past infection, most of these infections are asymptomatic with little evidence that the infection causes AVH. There are accumulating reports which suggest potential risk of HEV transmission by blood transfusion. However, detection of serological markers for HEV infection or HEV RNA in Egyptian blood banks is not routinely performed. 760 blood samples from apparently healthy donors at the National blood bank were tested for markers of acute HEV infection to estimate the seroprevalence of acute HEV infection, and potential risk of infection by blood transfusion. They included 124 females (16.82%) and 636 males (83.68%), with a mean age of 23.8 +/- 5.3 years and mean ALT value of 23.3 +/- 13.2 IU/ml. Samples were tested as pools of 10 subjects. Pools with highest reactivity were retested individually to determine the frequency of positive subjects. Out of the 760 samples, three (0.45%) samples were positive for anti-HEV IgM and two of them had HEV RNA as determined by RT-PCR. In conclusion, this study suggests that the tested blood donors have low prevalence of ongoing subclinical infection with HEV and that the potential risk of transfusion may be low.

Risk factors and immune response to hepatitis E viral infection among acute hepatitis patients in Assiut, Egypt.

Hepatitis E virus (HEV) infection is a common cause of acute viral hepatitis (AVH) in Egypt. We aimed to identify risk factors of HEV among acute hepatitis cases, measure HEV specific immune response to differentiate between symptomatic and asymptomatic infections. The study included symptomatic acute hepatitis (AH) patients (n = 235) and asymptomatic contacts (n = 200) to HEV cases. They completed a lifestyle questionnaire, screened for common hepatotropic viruses. Blood and serum samples were collected from patients and contacts after onset of disease and follow-up samples collected until convalescence. PBMC were separated and tested for specific HEV T-cell response by INF-gamma ELISPOT assay. Serum samples were tested for IgM and IgG anti-hepatitis E virus by ELISA. IgM antibodies to HAV were detected in 19 patients (8.1%), 37 (15.7%) with HBV, 10 (4.3%) with HCV. HEV infection was identified in 42 (16%) patients with AVH. Of the 200 contacts, 14 (7%) had serological evidence of recent HEV asymptomatic infection, showed stronger CMI responses than HEV infected subjects (2540 +/- 28 and 182 +/- 389 ISCs/106 cells, respectively; P < 0.05). In conclusion, HEV is a major cause of AVH in Egypt. Asymptomatic HEV patients are likely to have stronger immune responses including CMI responses, than symptomatic cases.

Schistosoma infection inhibits cellular immune responses to core HCV peptides.

Patients coinfected with hepatitis C virus (HCV) and the trematode, Schistosoma mansoni, have an increased incidence of viral persistence and accelerated fibrosis. To investigate immunological mechanisms responsible for this more aggressive natural history of HCV, the core HCV-specific T-cell responses were analysed in 44 donated blood units rejected because they had antibodies to HCV (anti-HCV). Half also had anti-S. mansoni antibodies, evidence of past or active infection. HCV-specific ELISPOT responses were examined using pools of 180 overlapping 9-mer peptides with offsets of one covering the core of HCV genotype 4a. Comparison of T-cell responses in blood units positive for both anti-HCV and anti-Schistosoma antibodies with blood units positive only for anti-HCV antibodies showed a significant decrease in core-specific T-cell IFN-gamma (505+/- 46 vs. 803 +/- 66 ISC/10(6) cells, P < 0.001), IL-4 (2 +/- 108 vs. 641 +/- 131 ISC/10(6) cells, P < 0.001), and IL-10 (159 +/- 105 vs. 466 +/- 407 ISC/10(6) cells, P < 0.002) responses. In contrast, there was no significant difference in cell-mediated immune response (CMI) to PHA mitogen between these two groups. Therefore, we concluded T cells from persons with anti-Schistosoma have reduced IFN-gamma, IL-4, and IL-10 secreting HCV-specific T-cell responses. This may explain why Schistosoma coinfection increases persistence and severity of HCV infection.

Vaccination with a Shigella DNA vaccine vector induces antigen-specific CD8(+) T cells and antiviral protective immunity.

A prototype Shigella human immunodeficiency virus type 1 (HIV-1) gp120 DNA vaccine vector was constructed and evaluated for immunogenicity in a murine model. For comparative purposes, mice were also vaccinated with a vaccinia virus-env (vaccinia-env) vector or the gp120 DNA vaccine alone. Enumeration of the CD8(+)-T-cell responses to gp120 after vaccination using a gamma interferon enzyme-linked spot assay revealed that a single intranasal dose of the Shigella HIV-1 gp120 DNA vaccine vector elicited a CD8(+) T-cell response to gp120, the magnitude of which was comparable to the sizes of the analogous responses to gp120 that developed in mice vaccinated intraperitoneally with the vaccinia-env vector or intramuscularly with the gp120 DNA vaccine. In addition, a single dose of the Shigella gp120 DNA vaccine vector afforded significant protection against a vaccinia-env challenge. Moreover, the number of vaccinia-env PFU recovered in mice vaccinated intranasally with the Shigella vector was about fivefold less than the number recovered from mice vaccinated intramuscularly with the gp120 DNA vaccine. Since the Shigella vector did not express detectable levels of gp120, this report confirms that Shigella vectors are capable of delivering passenger DNA vaccines to host cells and inducing robust CD8(+) T-cell responses to antigens expressed by the DNA vaccines. Furthermore, to our knowledge, this is the first documentation of antiviral protective immunity following vaccination with a live Shigella DNA vaccine vector.

IL-12 regulation of parasite antigen-driven IgE production in human helminth infections.

Recombinant IL-12 inhibits IgE synthesis by IL-4-stimulated lymphocytes from healthy persons and influences the development of Th subset selection involved in Ig isotype selection. Whether endogenous IL-12 production modulates IgE synthesis in patients with elevated serum IgE is unknown. To examine this question we studied the effects of neutralizing anti-IL-12 or recombinant IL-12 on parasite Ag-driven polyclonal IgE production and corresponding IFN-gamma and IL-4 synthesis by PBMC from helminth-infected individuals. The addition of neutralizing anti-IL-12 Ab significantly inhibited parasite Ag-driven IgE production in 11 of 12 individuals (p < 0.001). Recombinant IL-12 (1-10 U/ml) suppressed Ag-driven IgE production in a dose-dependent fashion up to 94% relative to untreated cultures. The effect of endogenous IL-12 on IgE production occurred, in part, by suppressing Ag-induced IL-4 and augmenting IFN-gamma production. IL-12 did not suppress IgE synthesis by purified B cells. An IFN-gamma-independent effect of IL-12 on Ag-driven IgE production was suggested by the ability of human rIL-12 to suppress IgE synthesis in the presence of neutralizing anti-IFN-gamma. This study demonstrates that IL-12 modulates helminth Ag-driven IgE production, in part, by regulating the relative quantities of IFN-gamma and IL-4 generated by Ag-specific lymphocytes.

Recent advances with recombinant bacterial vaccine vectors.

Bacille Calmette-Guerin (BCG), Listeria monocytogenes, Salmonellae and Shigellae have shown promise as vaccine vectors in experimental animal models. Although disappointing results in humans and non-human primates stalled the development of this vaccination strategy, interest in this approach was reinvigorated recently by the development of bacterial DNA-vaccine-vectors. The purpose of this review is to highlight the strengths and weaknesses of bacterial vaccine vectors, and to discuss the future prospects of these vaccine delivery systems.

Characterization of the binding of radioiodinated hybrid recombinant IFN-alpha A/D to murine and human lymphoid cell lines.

The hybrid recombinant human interferon (IFN) rIFN-alpha A/D was radioiodinated. Specific binding of [125I]rIFN-alpha A/D was observed with both human and murine cell lines. The binding of [125I]rIFN-alpha A/D to human Daudi cells had similar characteristics to the previously described binding of [125I]rIFN-alpha A or -alpha 2. The following lines of evidence demonstrated that [125I]rIFN-alpha A/D bound with high affinity to the same receptor on murine cells as murine IFN-alpha and -beta: (i) the binding of [125I]rIFN-alpha A/D to murine LBRM cells was inhibited to a similar extent by natural murine IFN-alpha, natural murine IFN-beta, and rIFN-A/D; (ii) the Kd (approximately 2 X 10(-10) M) obtained from both competition experiments and saturation binding experiments with [125I]rIFN-alpha A/D was comparable to the previously reported Kd for the binding of natural murine IFN-alpha and -beta to other murine cell lines; (iii) the size of the cross-linked [125I]rIFN-alpha A/D receptor complex formed on murine LBRM cells was similar to the previously reported cross-linked complex formed after binding radioiodinated natural murine IFN-beta to other murine cell lines. Due to the current lack of readily available recombinant murine IFN-alpha or -beta for radiolabeling and the previously demonstrated biological activity of rIFN-alpha A/D on murine cells, [125I]rIFN-alpha A/D should prove to be a useful reagent for further studies of murine IFN receptors.

Mucosal and systemic HIV-1 Env-specific CD8(+) T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector.

CD8(+) T-cell responses provide beneficial antiviral immunity against human immunodeficiency virus 1 (HIV-1). In this study, we show that intragastric vaccination with a Salmonella HIV-1 Env DNA vaccine vector generates Env-specific CD8(+) T-cells, both in mucosal and systemic lymphoid tissue. By contrast, intramuscular vaccination with the Env DNA vaccine alone only induced systemic CD8(+) T-cells. To our knowledge, this is the first report showing both mucosal and systemic CD8(+) T-cell responses following vaccination with a Salmonella vaccine vector. These data suggest that this mode of HIV-1 DNA vaccine delivery will be advantageous over parenterally administered HIV-1 DNA vaccines.

Immunogenicity of DNA vaccines that direct the coincident expression of the 120 kDa glycoprotein of human immunodeficiency virus and the catalytic domain of cholera toxin.

Passive antibody studies unequivocally demonstrate that sterilizing immunity against lentiviruses is obtainable through humoral mechanisms. In this regard, DNA vaccines represent an inexpensive alternative to subunit vaccine for mass vaccination programs designed to induce such responses to human immunodeficiency virus type I (HIV-1). At present, however, this vaccine modality has proven relatively ineffective at inducing humoral responses. In this report, we describe the immunogenicity of DNA vaccines that direct the coincident expression of the cholera toxin catalytic domain (CTA1) with that of the human immunodeficiency virus type I gp120 through genes either encoded in individual plasmids or in a single dicistronic plasmid. In BALB/cJ mice, coincident expression of CTA1 in either a separate plasmid or in the dicistronic plasmid in the DNA vaccines induced serum IgG responses to gp120 that were at least 1000-fold greater, and remained elevated longer than, the analogous responses in mice vaccinated with a DNA vaccine that expressed gp120 alone. In addition, mice vaccinated with CTA1 and gp120 produced significantly more gp120-specific IFN-gamma ELISPOTs than mice vaccinated with the gp120 DNA vaccine. Combined, these data show that the adjuvant properties of cholera toxin can be harnessed in DNA vaccine modalities.

Hepatitis C virus replication kinetics in chimpanzees with self-limited and chronic infections.

The availability of molecular beacon-based, real time polymerase chain reaction (PCR) and a semi-automated sample extraction procedure have made it possible for us to retrospectively examine HCV replication kinetics in HCV naive chimpanzees infected during the past 20 years. We compared these in 17 animals that developed chronic infection, and in 21 that developed self-limited infection. No differences were found in infecting dose, or replication kinetics in the acute phase between these two types of infection. An unanticipated finding was the fact that 10 of 17 animals developing chronic infection partially controlled virus replication for 48 +/- 48 weeks after typical acute phase viraemia, and prior to development of chronic infection. Twenty-nine out of 30 (29/30) sera, which were negative by quantitative PCR during the downregulated period, were, however, positive by the more sensitive Genprobe isothermal transcription-mediated amplification (TMA) assay. Thus, downregulation was not complete. Ten animals showing self-limited infection showed complete resolution of viraemia by TMA assay. Quasispecies analysis revealed that in all, except one case, the virus reappearing after downregulation was essentially identical to that of the originally infecting virus.