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The use of adjuvants capable of improving the deficient immune response to hepatitis B virus (HBV) vaccine in haemodialysis patients is highly needed. Among potential adjuvants, type I interferons deserve a special attention in view of their known effects promoting cellular and humoral immune responses. The aim of the present trial was to evaluate the effects of recombinant interferon-alpha2b (IFN) administered as an adjuvant of HBV vaccine in unvaccinated haemodialysis patients. A significant and early enhancing effect on the antibody response was observed in patients receiving IFN. In addition, a predominance of IgG1 anti-HBs along with a transient normalization of circulating Th1 lymphocytes was only found in patients receiving IFN who achieved an early seroprotection. However, 6 months after the last vaccine dose, no significant differences were observed in the seroprotection rate achieved in patients vaccinated with IFN compared to that in patients receiving HBV vaccine alone. Mild to moderate fever, asthenia, and arthromyalgia were the most common reactions that occurred in vaccinees given IFN. In conclusion, addition of IFN to HBV vaccine, under the conditions used in this trial, is safe and achieves an earlier and higher seroprotection rate improving Th1-dependent immune response in haemodialysis patients.

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.

HBV vaccine needs 3 injections over 6 months to induce immunity. Thus, the use of adjuvants capable of inducing earlier immune protection would be highly desirable. Most adjuvants may act by inducing cytokines, and among them, type I interferons (IFNs), deserve a special attention in view of the potent immunomostimulatory activity observed in mouse models and on dendritic cell functions. The aim of the present trial was to evaluate the effects of IFN-alpha administered as an adjuvant of HBV vaccine in healthy unvaccinated individuals. No significant enhancing effect on the antibody response was observed, in spite of an early and transient upregulation of costimulatory molecule expression on peripheral blood mononuclear cells, which may be suggestive of an IFN-mediated activation of antigen presenting cells. We conclude that, under the conditions used in this trial, natural IFN-alpha does not act as an adjuvant of the HBV vaccine in healthy unvaccinated individuals.

Avian influenza virus (H5N1) can be transmitted to humans, resulting in a severe or fatal disease. The aim of this study was to evaluate the immune cross-reactivity between human and avian influenza (H5N1) strains in healthy donors vaccinated for seasonal influenza A (H1N1)/(H3N2). A small frequency of CD4 T cells specific for subtype H5N1 was detected in several persons at baseline, and seasonal vaccine administration enhanced the frequency of such reactive CD4 T cells. We also observed that seasonal vaccination is able to raise neutralizing immunity against influenza (H5N1) in a large number of donors. No correlation between influenza-specific CD4 T cells and humoral responses was observed. N1 may possibly be a target for both cellular and humoral cross-type immunity, but additional experiments are needed to clarify this point. These findings highlight the possibility of boosting cross-type cellular and humoral immunity against highly pathogenic avian influenza A virus subtype H5N1 by seasonal influenza vaccination.

Background: The occurrence of, and risk factors for, HHV-8 infection have yet to be definitively determined, particularly among heterosexual individuals with at-risk behavior for sexually transmitted infections (STI). The objective of this study was to estimate the incidence and determinants of HHV-8 infection among HIV-uninfected individuals repeatedly attending an urban STI clinic., Methods: Sera from consecutive HIV-uninfected individuals repeatedly tested for HIV-1 antibodies were additionally tested for HHV-8 antibodies using an immunofluorescence assay. To identify determinants of HHV-8 infection, a nested case-control study and multivariate logistic regression analysis were performed., Results: Sera from 456 HIV-uninfected individuals (224 multiple-partner heterosexuals and 232 men who have sex with men (MSM]) were identified for inclusion in the study. The HHV-8 seroprevalence at enrollment was 9.4% (21/224; 95% C.I.: 6.0-14.2%) among heterosexuals with multiple partners and 22.0% (51/232; 95% C.I.: 16.9-28.0%) among MSM. Among the 203 multiple-partner heterosexuals and 181 MSM who were initially HHV-8-negative, 17 (IR = 3.0/100 p-y, 95% C.I.: 1.9 - 4.8) and 21 (IR = 3.3/100 p-y, 95% C.I:.2.1 - 5.1) seroconversions occurred, respectively. HHV-8 seroconversion tended to be associated with a high number of sexual partners during the follow-up among MSM (> 10 partners: AOR = 3.32 95% CI:0.89-12.46) and among the multiple-partner heterosexuals (> 10 partner; AOR = 3.46, 95% CI:0.42-28.2). Moreover, among MSM, HHV-8 seroconversion tended to be associated with STI (AOR = 1.80 95%CI: 0.52-7.96). During the study period the HIV-1 incidence was lower than that of HHV-8 among both groups (0.89/100 p-y among MSM and 0.95/100 p-y among multiple-partner heterosexuals)., Conclusion: The large difference between the incidence of HHV-8 and the incidence of HIV-1 and other STIs may suggest that the circulation of HHV-8 is sustained by practices other than classical at-risk sexual behavior.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. A large outbreak of CHIKV disease occurred in 2005 in the Indian Ocean Islands. Many cases have been imported in European countries. Laboratory confirmation of suspected cases is mandatory for control measures during an outbreak. We report a novel, real-time, reverse transcription-polymerase chain reaction (RT-PCR) for the nonstructural protein 1 region that can quantify a wide range of viral RNA concentrations. This assay was validated by in vitro experiments in which interferon-alpha, a well-known virus inhibitor, showed a dose-dependent inhibition of virus replication on Vero cells that was assessed by viral infectivity and viral RNA production. This new real-time RT-PCR was used to measure viral load in serum samples from cases recently imported to Italy, and may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication in patients.

Unlabelled: Severe acute respiratory syndrome (SARS) is a newly emerging, highly transmissible and fatal disease caused by a previously unknown coronavirus (SARS-CoV). Existing in non-identified animal reservoirs, SARS-CoV continues to represent a threat to humans because there is no effective specific antiviral therapy for coronavirus infections., Objectives: Starting from the observation that cyclopentenone cyclooxygenase (COX) metabolites are active against several RNA viruses, we investigated the effect of the COX inhibitor indomethacin on coronavirus replication., Methods: Work involving infectious SARS-CoV was performed in biosafety level 3 facilities. SARS-CoV was grown in monkey VERO cells and human lung epithelial A549 cells, while canine coronavirus (CCoV) was grown in A72 canine cells. Antiviral activity was analysed by determining infective virus titres by TCID50, viral RNA synthesis by Northern blot analysis and real-time RT-PCR, and viral protein synthesis by SDS-PAGE analysis after 35S-methionine-labelling. Antiviral efficacy in vivo was determined by evaluating virus titres in CCoV-infected dogs treated orally with 1 mg/kg body weight indomethacin (INDO)., Results: Unexpectedly, we found that INDO has a potent direct antiviral activity against the coronaviruses SARS-CoV and CCoV. INDO does not affect coronavirus binding or entry into host cells, but acts by blocking viral RNA synthesis at cytoprotective doses. This effect is independent of cyclooxygenase inhibition. INDO's potent antiviral activity (>1,000-fold reduction in virus yield) was confirmed in vivo in CCoV-infected dogs., Conclusions: The results identify INDO as a potent inhibitor of coronavirus replication and suggest that, having both anti-inflammatory and antiviral activity, INDO could be beneficial in SARS therapy.

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.

Background: Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV, characterized by an over-exuberant immune response with lung lymphomononuclear cells infiltration and proliferation that may account for tissue damage more than the direct effect of viral replication. This study is aimed at investigating the capability of SARS-CoV to activate IFN-alpha and -gamma expression in lymphomonocytes (PBMC) from healthy donors, evaluating whether viral replication is necessary for this activation., Results: SARS-CoV virus is able to induce both IFN-alpha and -gamma mRNA accumulation and protein release in a dose-dependent manner, MOI 10 being the most effective. The time course curve indicated that IFN-alpha mRNA induction peaked at 24 h.p.i,. whereas IFN-gamma mRNA was still increasing at 48 h.p.i. Released IFN (both types) reached a plateau after 24-48 h.p.i. and remained rather stable over a 5-day period. A transient peak of negative strand viral RNA was detected after 1-2 days of infection, but neither infectious virus progeny yield nor newly produced viral genomic RNA could be evidenced in infected cultures, even after prolonged observation time (up to 13 days). Cocultivation of PBMC with fixed SARS-CoV-infected Vero cells was even more efficient than exposure to live virus in eliciting IFN-alpha and -gamma induction. A combination of IFN-alpha and -gamma strongly inhibited SARS-CoV replication in Vero cells, while the single cytokines were much less effective., Conclusions: This study provides evidence that SARS-CoV is able to induce in normal PBMC a coordinate induction of IFN-alpha and -gamma gene expression. Virus replication is not necessary for IFN induction since efficient IFN expression could be obtained also by the cocultivation of normal PBMC with fixed SARS-CoV-infected cells. Concomitant activation of IFN-alpha and -gamma gene expression by SARS-CoV in vivo may be relevant for the pathogenesis of the disease, both for the possible involvement in immunomediated damage of the tissues and for the strong inhibition of SARS-CoV replication as a result of combined cytokine action.

Objective: To investigate the prevalence of human herpesvirus 8 (HHV-8; Kaposi sarcoma-associated herpesvirus) infection in patients with lymphoproliferative skin diseases such as large-plaque parapsoriasis (LPP) and mycosis fungoides compared with inflammatory cutaneous conditions or healthy control subjects., Design: A survey study was undertaken in 123 subjects with various clinical conditions., Setting: All patients had been seen in the Dermatology Department of the San Gallicano Dermatology Institute, Rome, Italy, in the last 2 years., Patients: Forty-five patients with inflammatory or autoimmune cutaneous diseases, 50 healthy control subjects, 10 patients with LPP, 12 patients with mycosis fungoides, and 6 patients with classic Kaposi sarcoma were included in the study., Main Outcome Measures: The prevalence of HHV-8 infection was investigated with serologic studies using the gold standard assay based on body cavity-based B-cell lymphoma-1 cells latently infected with HHV-8. The presence of HHV-8 conserved sequence, corresponding to open reading frame 26, was also assessed in the peripheral blood and lesion tissue samples from patients with lymphoproliferative cutaneous diseases with nested polymerase chain reaction. The presence and distribution of cell types infected with HHV-8 in the lesion tissues was determined with immunohistochemical staining with the monoclonal antibody directed against the latent nuclear antigen-1 of HHV-8 encoded by open reading frame 73., Results: In healthy control subjects and patients with inflammatory skin diseases, 13.9% were found to have antibody against HHV-8, consistent with the seroprevalence population in Italy. A highly significant association of HHV-8 infection and LPP was found (100%) compared with mycosis fungoides (25%). The peripheral blood mononuclear cells in 8 of 10 patients with LPP were found to harbor viral sequences at nested polymerase chain reaction, whereas none of them had a detectable serum viral load. All LPP lesion tissue samples were positive for HHV-8-encoded open reading frame 26, and the presence of HHV-8-infected cells was confirmed by immunohistochemistry profiles performed on paraffin-embedded tissues from 4 of 10 patients. The positive cell types included endothelial cells and the infiltrating dermal lymphocytes, characteristic hallmarks of LPP. Analysis of T-cell receptor gamma chain rearrangements in lesion tissue from our patients confirmed the lack of a significant association between T-cell clonality and LPP., Conclusion: These data suggest that HHV-8 may play a role in the onset of LPP, a disease whose cause and evolution are still undefined and which has often been considered the early stage of mycosis fungoides.

In the search for new strategies against HIV-1 and on the basis of a number of previous studies reporting on the capacity of certain polyanionic compounds to influence the replication of HIV-1, we prepared a few chemically oversulfated dermatan and chondroitin sulfates. Four of these compounds and two samples of heparin were bound to activated Sepharose through either their carboxylic groups, or their aldehydic groups, or their deacetylated primary amino groups. Some of these so-derivatised resins, packed into columns, proved able to remove HIV-1 IIIB, a laboratory adapted strain, and one clinical primary isolate from an AIDS patient, from infected cell culture medium. The resins bind the virus very tightly and could be useful for capturing the virus from infected fluids.

Orthopoxvirus zoonosis or smallpox as result of bioterrorism or biological warfare represents a risk for epidemic spread. By monitoring T-cell responses by flow cytometry, we observed a recall response after recent vaccination against smallpox. When the high similarity between the orthopoxviruses is considered, this rapid assay that uses vaccinia antigens could identify recently exposures.

IFN alpha causes a modest reduction of HIV-1 expression in chronically infected monocytoid U937 cells. However, the ratio between cell-associated and shed viral p24 antigen is altered, being the cell-associated fraction dose-dependently enhanced by IFN. Furthermore, a significant decrease of infectivity of both cell-associated and shed material is observed. Transmission electron microscopy of IFN-treated cells revealed virus assembly being strongly inhibited, with the production of morphologically altered (tear-drop shaped) virus particles. Proteolytic processing of gag proteins appeared to be normal in IFN-treated cultures. However, virions shed from IFN-treated cells showed a markedly reduced incorporation of virus-specific gp120 and cell-derived ICAM-1 by the virus envelope. Additionally, these particles showed a significantly decreased ability to become bound to CD4+ target cells, accounting for, at least in part, the observed decrease of infectivity. Taken together, the data suggest that, in chronically infected cells, IFN alpha can affect late stages of HIV-1 replication, by inhibiting virus assembly and release, and by reducing the infectivity of shed virions. The latter effect seems to be due, at least in part, to altered incorporation of surface glycoproteins and defective particle formation. The relationship between impaired gp120 incorporation and altered morphogenesis of HIV-1 virions is under investigation.

We have investigated the mechanism of interferon (IFN) induction in peripheral blood mononuclear cells by HIV-1(IIIB)-infected H9 cells or by recombinant gp120. A monoclonal antibody specific for the galactosylsphingosinyl moiety in galactocerebrosides and sulfatides inhibited IFN induction in a dose-dependent manner. Furthermore, exogenous sulfatides inhibited with an ID50 of approximately 1 microM, whereas galactocerebrosides were not inhibitory at 40 times higher concentrations. These studies suggest that sulfate containing galactolipids such as sulfatides on responder cells may be part of the gp120-membrane complex that initiates the induction of IFN. A partial homology of an epitope on the V3 loop of gp 120 with a previously suggested binding domain for sulfated glycoconjugates supports this conclusion.

Cellular adhesion molecules, such as ICAM-1, -2, and -3; LFA-1; and HLA class I and II are incorporated into HIV-1 virions during budding from infected cells. These virion-associated molecules can be involved in the adsorption to susceptible cells displaying the corresponding counterligands. A number of cytokines have been shown to upregulate the cellular expression of adhesion molecules, such as ICAM-1 and HLA-DR. In this study we investigated the effects of IFN-gamma on the incorporation of ICAM-1, LFA-1, and HLA-DR into mature HIV-1 progeny from chronically infected cells. The ability of such virus progeny to infect either CD4-positive or -negative cells was also investigated. The results indicate that IFN-gamma stimulates the expression of ICAM-1 and of HLA-DR on HIV-1-infected cells, whereas LFA-1 expression is unaffected. The same modifications were also observed on virus progeny, because specific MAbs to ICAM-1 and HLA-DR captured infectious HIV-1 from IFN-treated cells with higher efficiency as compared to virus from control cells, whereas virus binding to anti LFA-1 MAb was unchanged. Moreover, the HIV-1 progeny released from IFN-treated cells showed an increased ability to bind to and to infect CD4-negative cells, whereas the infectivity was basically unchanged for CD4-positive cells. Our results suggest that cytokines, as well as other soluble factors, may expand the host cell range of HIV-1, possibly through modifications of the cell-derived surface molecules on the virions.(ABSTRACT TRUNCATED AT 250 WORDS)

Objectives: To explore the possibility that HIV-1 budding and cellular adhesion molecules co-polarize at cell-to-cell contact sites. To investigate the incorporation of host-cell-derived adhesion molecules into HIV-1., Methods: The cellular sites involved in HIV-1 budding were examined by transmission electron microscopy. Single and double immunocytochemistry staining was used to evaluate the cellular distribution of the viral matrix protein and adhesion molecules. Quantitative flow cytometry was used to measure the cellular expression of adhesion molecules. An immunocapture technique was used to measure the presence of cell-derived proteins on HIV-1. The captured virus was measured by a p24 antigen assay. The infectivity of virus captured by monoclonal antibodies was tested by measuring the virus antigen yield in supernatants after the addition of sensitive cells., Results: Released and budding HIV-1 was mainly localized at the cell-to-cell contact regions. This feature was consistent with a polarized staining for the virus matrix protein p18 at cell-to-cell contact regions. Intercellular adhesion molecules (ICAM)-1 in HIV-1-infected cells were polarized on both isolated cells and syncytia, co-localizing with HIV-1 matrix protein. HIV-1 incorporated all the adhesion molecules expressed by the host cells, although without quantitative correlation with their cellular expression., Conclusions: HIV-1 is released at cell-to-cell membrane contact sites. Both ICAM-1 and virus matrix protein co-polarized on isolated cells and syncytia at the sites involved in the recruitment of uninfected cells. The impressive concentration of ICAM at cell sites where most virions are released may account for the acquisition of these membrane proteins by the HIV-1 progeny, and may be important for the cell-mediated spread.

Human umbilical vein endothelial cells (HUVEC) can be abortively infected with HIV-1, but virus production is rescued by the addition of T cells. Monoclonal antibody (Mab) to ICAM-1, but not its Fab' fragment or MAbs to LFA-1 and PECAM-1, increases HIV-1 infection of HUVEC by enhancing HIV-1 absorption. Enhancement by anti ICAM-1 is probably due to a bridging effect different from the ADE mediated by anti-gp120 that involves FcR or CR-mediated capture of the virus-antibody complex. Since antibodies to cell membrane molecules are present in HIV-1 infected patients, the ADE mediated by such a mechanism can be important in AIDS pathogenesis.

HIV-1 acquires cell membrane proteins during budding. The cell membrane proteins (CMP) profile of laboratory HIV-1 strains grown in different host cells was established, by using an immobilized antibody capture (IAC), to verify whether CMPs present on HIV-1 correlate with its host cell origin. HIV-1 grown in different cell lines incorporates cell markers such as CD3, CD19, CD14, CD31 and IL 2-R, according to the distinctive expression of these antigens on the host cells. Furthermore, also T-tropic and monocytotropic HIV-1 strains display host cell specific markers, supporting the hypothesis that virus associated CMPs are a marker of host cell origin.

We have studied the mechanism of IFN induction by HIV-1 in peripheral blood mononuclear cells (PBMC), using recombinant viral membrane glycoproteins as potential inducers. Whereas 8 nM HIVIIIB-derived gp120 resulted in IFN levels between 80 and 2000 IU/ml with PBMC from different donors, gp120 from the MN strain was not an inducer. Preincubation of HIVIIIB-gp120 with a monoclonal antibody (mAB) to its CD4 binding domain or of PBMC with a mAB to the gp120 binding domain of CD4 abolished IFN induction. Antibodies against the third extracellular domain of CD4 which did not block binding of gp120, however, were also inhibitory. Furthermore, several mABs to the third variable loop (V3) of HIVIIIB-gp120 also blocked IFN induction, suggesting an important role of V3 in this process. This was further supported by the inhibitory action of peptides homologous to complete or partial sequences of V3. We conclude that after binding of gp120 to its CD4 receptor the V3 loop can be positioned close to the membrane of the responder cells by bending of gp120-occupied CD4 at its hinge region between extracellular domains 2 and 3. As a result V3 is able to interact with a V3-specific "secondary receptor" on the membranes of these cells. We suggest that it is the latter interaction which triggers IFN induction.

Identification of cell-derived molecules on infectious human immunodeficiency virus type 1 (HIV-1) particles may be helpful in investigating mechanisms of HIV infection and in vaccine studies. Some of these molecules were detected on HIV-1 virions in previous studies, but rather elaborate methods were used. The method presented here allows an extensive characterization of the cell surface molecules associated with HIV-1 by capturing virus particles on monoclonal antibodies to cell membrane antigens bound to plastic wells. Binding of infectious virus was assessed by adding permissive target cells (C8166) and determining viral replication. With this procedure, beta 2-microglobulin, HLA-DR, intercellular adhesion molecule-1, and leukocyte function antigen-1 were found on HIV-1 particles from laboratory strains and primary clinical isolates. In contrast, CD19, CD4, and CD8 molecules were not detected.

Immunological abnormalities present in HIV-1-infected individuals often reflect an imbalance of cytokine production. The HIV-1 gp120 has the ability to induce a number of cytokines, and to enhance immunoglobulin release by normal peripheral blood mononuclear cells (PBMC) in vitro, in the absence of IL-2 production and of lymphoproliferation. This study provides evidence that gp120 is a potent IL-10 inducer in normal PBMC cultures. The pattern of other cytokines induced by gp120 includes interferon-alpha (IFN-alpha) and IFN-gamma, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-1 alpha and -beta, and not IL-2 and IL-4. These findings further define the pattern of cytokine release induced by gp120 on human resting PBMC. Furthermore, the present findings roughly parallel those observed both in the sera of patients and in the mononuclear cells from HIV+ individuals early after infection, suggesting that gp120 could be a good candidate as one of the agents responsible for cytokine dysregulation observed in HIV-1-infected individuals.

Similarly to HIV-infected cells, recombinant HIV-1 glycoprotein 120 induces acid-labile interferon production in peripheral blood mononuclear cells from healthy donors. Acid lability of this interferon is due to the presence of both IFN-alpha and -gamma molecules. In fact, although not revealed by neutralization of antiviral activity with antibody to IFN-gamma, the presence of IFN-gamma was shown both immunoenzymatically and by detection of specific mRNA in gp120-stimulated cells. The source of IFN-gamma appears to be a T cell present in the CD4-enriched subpopulation. Cultures treated with monoclonal antibodies to the ICAM-1 and LFA-1 adhesion molecules showed an impaired release of both IFN types after gp120 stimulation, suggesting a crucial role of cell-to-cell interactions in the process leading to IFN production. Our data suggest that the HIV envelope glycoprotein could be responsible for the induction of endogenous IFN-alpha and -gamma observed in AIDS patients.