Virus-specific Immune Responses Research Articles

Hepatitis C virus-specific cellular and humoral immune responses following immunization with a multi-epitope fusion protein

Hepatitis C virus (HCV) is an important causative agent of acute and chronic hepatitis worldwide. We prepared a fusion protein in the vector of pET-11d that included three conserved broadly neutralizing B-cell epitopes and a series of T-cell epitopes located in the HCV NS3 region. In vivo administration of this fusion construct resulted in specific CD8+ cytotoxic lymphocytes in both PBMCs and splenocytes that could recognize specific antigen sites that could be detected by FACS. An HCVcc system was established and applied to detect HCV-specific neutralizing antibodies. These results suggest that the multi-epitope fusion protein is immunogenic and can elicit both humoral and cellular immune responses. In particular, this fusion protein is able to elicit HCV-specific neutralizing antibodies, which are critical for viral clearance. This construct may be significant for vaccine development and could be a potential candidate to be included in the design of a prophylactic and therapeutic vaccine against HCV.

Alterations in Immune Function are Associated with Liver Enzyme Elevation in HIV and HCV Co-infection after Commencement of Combination Antiretroviral Therapy

The cause of liver enzyme elevation during combination antiretroviral therapy in people with human immunodeficiency virus and hepatitis C virus co-infection is unclear. We followed 12 subjects (five with alanine transaminase elevation) for 24 weeks after combination antiretroviral therapy commencement. Immune responses against hepatitis C virus, human immunodeficiency virus and other viruses were assessed by interferon-γ ELISpot. Plasma cytokines, chemokines and anti-hepatitis C virus antibody levels were measured. Those with liver enzyme elevation had higher ELISpot responses both against hepatitis C virus non-structural regions and other viral antigens, and their anti-hepatitis C virus antibody levels were consistently higher, suggesting that reconstitution of both hepatitis C virus-specific and non-hepatitis C virus-specific immune responses may be associated with liver transaminase elevation during combination antiretroviral therapy.

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.

High Cell-Free Virus Load and Robust Autologous Humoral Immune Responses in Breast Milk of Simian Immunodeficiency Virus-Infected African Green Monkeys

The design of immunologic interventions to prevent postnatal transmission of human immunodeficiency virus (HIV) will require identification of protective immune responses in this setting. Simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs), a species that develops an AIDS-like illness following experimental infection, transmit the virus at a high rate during breastfeeding. In contrast, postnatal transmission of SIV occurs rarely or not at all in natural, asymptomatic primate hosts of SIV. These contrasting transmission patterns provide a unique opportunity to study mechanisms that evolved to protect suckling infants from SIV infection. We compared the virologic and immunologic properties of milk of SIV-infected and uninfected natural hosts of SIV, African green monkeys (AGMs), to that of RMs. Interestingly, despite a low number of milk CD4(+) T lymphocytes in uninfected AGMs, milk virus RNA load in SIV-infected AGMs was comparable to that of SIV-infected RMs and that in AGM plasma. This observation is in contrast to the relatively low virus load in milk compared to that in plasma of SIV-infected RMs and HIV-infected women. Milk of SIV-infected AGMs also displayed robust virus-specific cellular immune responses. Importantly, an autologous challenge virus-specific neutralization response was detected in milk of five of six SIV-infected AGMs that was comparable in magnitude to that in plasma. In contrast, autologous challenge virus neutralization was not detectable in milk of SIV-infected RMs. The autologous virus-specific adaptive immune responses in breast milk of AGMs may contribute to impedance of virus transmission in the infant oral/gastrointestinal tract and the rarity of postnatal virus transmission in natural hosts of SIV.

Innate Immune Sensing of Retroviral Infection via Toll-like Receptor 7 Occurs upon Viral Entry

Innate immune sensors are required for induction of pathogen-specific immune responses. Retroviruses are notorious for their ability to evade immune defenses and establish long-term persistence in susceptible hosts. However, some infected animals are able to develop efficient virus-specific immune responses, and thus can be employed for identification of critical innate virus-sensing mechanisms. With mice from two inbred strains that control retroviruses via adaptive immune mechanisms, we found that of all steps in viral replication, the ability to enterthe host cell was sufficient to induce antivirus humoral immune responses. Virus sensing occurred in endosomes via a MyD88-Toll-like receptor 7-dependent mechanism and stimulated virus-neutralizing immunity independently of type I interferons. Thus, efficient adaptive immunity to retroviruses is induced invivo by innate sensing of the early stages of retroviral infection.

Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines

BackgroundMolecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection.ResultsFull length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array.ConclusionsOverall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent TH1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

Induction of immune responses in ducks with a DNA vaccine encoding duck plague virus glycoprotein C

BackgroundA DNA vaccine expressing glycoprotein C (gC) of duck plague virus (DPV) was evaluated for inducing immunity in ducks. The plasmid encoding gC of DPV was administered via intramuscular (IM) injection and gene gun bombardment.ResultsAfter immunization by both routes virus-specific serum antibody and T-cell responses developed. Vaccination of ducks by IM injection induced a stronger humoral, but weaker cell-mediated immune response. In contrast, a better cell-mediated immune response was achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis with as little as 6 μg of DNA.ConclusionsThis demonstrated that both routes of DNA inoculation can be used for eliciting virus-specific immune responses. Although DNA vaccine containing DPV gC is effective in both intramuscular injection and gene gun bombardment, the latter could induce significantly higher cell-mediated responses against DPV.

Flora against the flu

Commensal bacteria promote influenza virus-specific adaptive immune responses in an inflammasome-dependent manner.

Neutrophils sustain effective CD8+ T‐cell responses in the respiratory tract following influenza infection

Neutrophils have an important role in early host protection during influenza A virus infection. Their ability to modulate the virus-specific adaptive immune response is less clear. Here, we have used a mouse model to examine the impact of neutrophils on CD8(+) T-cell responses during influenza virus infection. CD8(+) T-cell priming, expansion, migration, cytokine secretion and cytotoxic capacity were investigated in the virus-infected airways and secondary lymphoid organs. To do this, we utilised a Ly6G-specific monoclonal antibody (mAb; 1A8) that specifically depletes neutrophils in vivo. Neutrophil depletion early after infection with influenza virus strain HKx31 (H3N2) did not alter influenza virus-derived antigen presentation or naïve CD8(+) T-cell expansion in the secondary lymphoid organs. Trafficking of virus-specific CD8(+) T cells into the infected pulmonary airways was also unaltered. Instead, early neutropenia reduced both the overall magnitude of influenza virus-specific CD8(+) T cells, together with impaired cytokine production and cytotoxic effector function. Therefore, neutrophils are important participants in anti-viral mechanisms that sustain effective CD8(+) T-cell responses in the respiratory tract of influenza virus-infected mice.

195 Hepatitis C Virus-Specific Immune Response Among Egyptian Healthcare Workers at High Risk of Infection Without Viremia or Seroconversion*

195 Hepatitis C Virus-Specific Immune Response Among Egyptian Healthcare Workers at High Risk of Infection Without Viremia or Seroconversion*

Intranasal immunization of porcine reproductive and respiratory syndrome virus entrapped biodegradable PLGA- nanoparticles elicits virus specific cross-protective immune responses in pigs (161.21)

Abstract Porcine reproductive and respiratory syndrome (PRRS) is an economically very important endemic chronic respiratory viral disease of pigs. Live PRRS virus (PRRSV) vaccines are not safe in pregnant sows and boars, and currently used killed vaccines are ineffective. In this study, killed PRRSV Ags were entrapped in poly-DL-lactide-co-glycolide (PLGA) nanoparticles (100-500nm by SEM) with an approximate 50% entrapment efficiency. Internalization and localization of nanoparticles-PRRSV Ags inside the endosomes of pig alveolar macrophages was demonstrated by confocal microscopy. Immune correlates of protection were analyzed in nanoparticles-PRRSV Ags immunized and genetically variant PRRSV challenged pigs, inoculated intranasally, at post-challenge day 15. In nanoparticles immunized pig lungs, reduced PRRSV load and reduced gross lung lesions were detected compared to pigs unimmunized or immunized with free killed PRRSV Ags and challenged. Immunologically, increased frequency of lymphocytes, CD4+, CD8+, γδ T cells, myeloid cells, and reduced frequency of Tregs were detected in lungs, blood, and tracheobronchial lymph nodes. In addition, increased secretion of cytokines, IFN-γ, IL-6, and reduced IL-10 and TGF-β by ex vivo restimulated mucosal immune cells was detected. Thus, our results suggested that mucosal delivery of nanoparticles- PRRSV killed Ags has the potential to control PRRS.

Enhanced HIV-1 Gag-specific immunity induced by a sPD-1-based vaccine directed to dendritic cells (53.23)

Abstract HIV/AIDS is a serious disease worldwide. The development of a safe, effective and affordable HIV-1 vaccine remains the ultimate solution. This study is based on the discovery of a negative immune regulatory system, the PD-1/PD-L pathway, which exists on both human and other animals. The PD-1 ligands (PD-L1 and PD-L2) are expressed on DCs. So we hypothesize to utilize the soluble PD-1 parts to intervene in the negative signal transmission of the PD-1 pathway and to target the antigen to DCs as a mean to indentify vaccine strategy to enhance the virus specific immune responses. Here we constructed the novel vaccine and controls by inserting soluble PD-1 gene and/or HIV-1 p24 gene into the pVAX1 vector with fusing of rabbit Fc fragment named mspd1-p24-fc, and controls named mspd1-IgVΔ-p24-fc and p24-fc. When compared with control vaccines, we discovered that mspd1-p24fc can significantly enhance HIV-1 Gag-specific immune responses by measuring the number of IFN-γ expressed CD4 and CD8 T cells using Elispot assays and CD8 T cell tetramer staining. Furthermore, the mspd1-p24-fc can significantly enhance humoral immune responses by anti-Gag antibody titer analysis. Importantly, this novel vaccine protects mice against recombinant vaccinia virus-gagpol challenges. This novel vaccine design may be used as DNA vaccine model against other infectious disease and cancer which need eliciting significant antigen specific humoral and cellular immune responses.

Evolution of Hepatitis B and C serum markers: a still challenging issue

Evolution of Hepatitis B and C serum markers: a still challenging issue

Fever with Thrombocytopenia Associated with a Novel Bunyavirus in China

Heightened surveillance of acute febrile illness in China since 2009 has led to the identification of a severe fever with thrombocytopenia syndrome (SFTS) with an unknown cause. Infection with Anaplasma phagocytophilum has been suggested as a cause, but the pathogen has not been detected in most patients on laboratory testing. We obtained blood samples from patients with the case definition of SFTS in six provinces in China. The blood samples were used to isolate the causal pathogen by inoculation of cell culture and for detection of viral RNA on polymerase-chain-reaction assay. The pathogen was characterized on electron microscopy and nucleic acid sequencing. We used enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and neutralization testing to analyze the level of virus-specific antibody in patients' serum samples. We isolated a novel virus, designated SFTS bunyavirus, from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. Electron-microscopical examination revealed virions with the morphologic characteristics of a bunyavirus. The presence of the virus was confirmed in 171 patients with SFTS from six provinces by detection of viral RNA, specific antibodies to the virus in blood, or both. Serologic assays showed a virus-specific immune response in all 35 pairs of serum samples collected from patients during the acute and convalescent phases of the illness. A novel phlebovirus was identified in patients with a life-threatening illness associated with fever and thrombocytopenia in China. (Funded by the China Mega-Project for Infectious Diseases and others.).

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

Partial Regulatory T Cell Depletion Prior to Acute Feline Immunodeficiency Virus Infection Does Not Alter Disease Pathogenesis

Feline immunodeficiency virus (FIV) infection in cats follows a disease course similar to HIV-1, including a short acute phase characterized by high viremia, and a prolonged asymptomatic phase characterized by low viremia and generalized immune dysfunction. CD4+CD25hiFoxP3+ immunosuppressive regulatory T (Treg) cells have been implicated as a possible cause of immune dysfunction during FIV and HIV-1 infection, as they are capable of modulating virus-specific and inflammatory immune responses. Additionally, the immunosuppressive capacity of feline Treg cells has been shown to be increased during FIV infection. We have previously shown that transient in vivo Treg cell depletion during asymptomatic FIV infection reveals FIV-specific immune responses suppressed by Treg cells. In this study, we sought to determine the immunological influence of Treg cells during acute FIV infection. We asked whether Treg cell depletion prior to infection with the highly pathogenic molecular clone FIV-C36 in cats could alter FIV pathogenesis. We report here that partial Treg cell depletion prior to FIV infection does not significantly change provirus, viremia, or CD4+ T cell levels in blood and lymphoid tissues during the acute phase of disease. The effects of anti-CD25 mAb treatment are truncated in cats acutely infected with FIV-C36 as compared to chronically infected cats or FIV-naïve cats, as Treg cell levels were heightened in all treatment groups included in the study within two weeks post-FIV infection. Our findings suggest that the influence of Treg cell suppression during FIV pathogenesis is most prominent after Treg cells are activated in the environment of established FIV infection.

Correlation between CD4+ T-cell loss and Gag-specific T cells in different intestinal sites of chronically SIV-infected rhesus monkeys

To determine the loss of CD4+ T cells and virus-specific cytotoxic T cells (CTL) in different mucosal sites of rhesus monkeys infected with simian immunodeficiency virus (SIV). A cross-sectional comparative investigation of seven different mucosal sites from chronically SIV-infected rhesus monkeys was performed by analyzing blood and mucosal lymphocytes. Mucosal lymphocytes were isolated from duodenum, jejunum, ileum and colon as well as from vagina, cervix and uterus of SIV-infected rhesus monkeys at necropsy. CD4+ T cells and SIV-Gag-specific CTL were determined in blood and mucosal samples by flow cytometry. A significant depletion of CD4+ T cells was observed in blood and all mucosal sites of SIV-infected rhesus monkeys compared to uninfected animals. But the mean percentage loss of CD4+ T cells varied between 66 and 95% between the different mucosal tissues. The frequency of CTL ranged between 0.4 and 2.4% with the highest proportions in vagina and cervix. Among the intestinal sites the mean levels of CTL correlated with mean percentage loss of CD4+ T cells. A discriminative pronounced loss of CD4+ T cells among the mucosal tissues confirmed that viral replication affects different mucosal sites in a distinct way. Despite high levels of CTL, especially in vagina and cervix, the severe loss of mucosal CD4+ T cells could not be prevented during chronic SIV infection. However, within the four sites of the intestine a high virus-specific cellular immune response correlated with better preservation of CD4+ T cells.

Antiretroviral Therapy-Induced Dominant Interleukin-2 HIV-1 Gag CD4+ T Cell Response: Evidence of Functional Recovery of HIV-1-Specific CD4+ T Cells

Prolonged antiretroviral treatment (ART) significantly changes the cytokine secretion capacities of HIV-1-specific T cells. However, it is unclear whether these changes result from decreased viremia or they correspond to true functional recovery of viral-specific immune response. To study this issue, we analysed the quantitative and qualitative differences of HIV-1-specific and polyclonal CD4+ and CD8+ T cells between 26 naive and 52 treated individuals. HIV-1 Gag and staphylococcal enterotoxin B (SEB)-reactive T cells were determined by flowcytometric intracellular secretion of IFN-γ or/and ΙL-2. ART resulted in increase of single IL-2 and decrease of single IFN-γ-secreting HIV-1 CD4+ T cells, while both cytokines secreting HIV-1 CD4+ T cells were presented in comparable frequencies in both groups. Viral loads correlated negatively with single IL-2 and positively with single IFN-γ-secreting HIV-1 CD4+ cells. Single IL-2 HIV-1 CD4+ T cells correlated positively with both cytokines secreting polyclonal CD8+ T cells. By qualitative analysis, a dominant IL-2 HIV-1 CD4+ T cell response (> 70% single IL-2) was identified only in ART suppressed patients, who also generated increased dual specific polyclonal CD8+ T cells. Polyfunctional HIV-1 CD4+ T cell responses were detected even in naive individuals with high viremia. In conclusion, the presence of dominant IL-2 HIV-1 CD4+ T cell response, associated with increased CD8+ T cells capable to produce IL-2, indicates that the recovery of HIV-1-specific CD4+ T cell functionality under ART is a feasible goal. Furthermore, polyfunctional HIV-1 CD4+ T cell responses seem not to be directly involved in viral replication control.

Human papillomavirus 16 E7 protein inhibits interferon‐γ‐mediated enhancement of keratinocyte antigen processing and T‐cell lysis

Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ineffective virus-specific immune response, and prolonged infection promotes anogenital cancer. To determine whether poor antigen presentation by HPV-infected keratinocytes (KCs) contributes to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein (E7-KCs) were compared for susceptibility to T-cell-mediated lysis directed to ovalbumin (OVA) processed for presentation by the KCs. Interferon (IFN)-γ efficiently enhanced susceptibility to lysis of KCs presenting OVA, but not of E7-KCs similarly presenting OVA. E7-KCs also exhibited impaired IFN-γ-induced upregulation of transcription of major histocompatibility complex class I antigen processing and presentation-associated genes, and of membrane SIINFEKL-H-2K(b) complexes. Thus, expression of HPV 16 E7 protein in KCs may inhibit enhancement by IFN-γ of KC sensitivity to T-cell lysis, by impairing antigen presentation.

Persistence of virus-specific immune responses in the central nervous system of mice after West Nile virus infection.

BackgroundWest Nile virus (WNV) persists in humans and several animal models. We previously demonstrated that WNV persists in the central nervous system (CNS) of mice for up to 6 months post-inoculation. We hypothesized that the CNS immune response is ineffective in clearing the virus.ResultsImmunocompetent, adult mice were inoculated subcutaneously with WNV, and the CNS immune response was examined at 1, 2, 4, 8, 12 and 16 weeks post-inoculation (wpi). Characterization of lymphocyte phenotypes in the CNS revealed elevation of CD19+ B cells for 4 wpi, CD138 plasma cells at 12 wpi, and CD4+ and CD8+ T cells for at least 12 wpi. T cells recruited to the brain were activated, and regulatory T cells (Tregs) were present for at least 12 wpi. WNV-specific antibody secreting cells were detected in the brain from 2 to 16 wpi, and virus-specific CD8+ T cells directed against an immunodominant WNV epitope were detected in the brain from 1 to 16 wpi. Furthermore, these WNV-specific immune responses occurred in mice with and without acute clinical disease.ConclusionsVirus-specific immune cells persist in the CNS of mice after WNV infection for up to 16 wpi.