Interferon-gamma Secreting Cells Research Articles

Abstract 687: Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis

Abstract ES2B-C001 is a virus-like particle (VLP) vaccine against human HER-2, developed for the therapy of breast cancer. We show here that ES2B-C001 effectively inhibits mammary carcinoma growth and metastasis in a transgenic mouse model expressing activated human HER-2. ES2B-C001 vaccine is a tag/catcher conjugation system: Acinectobacter phage 205 (AP205) capsid VLP, each with 180 tag peptides, are conjugated with catcher-HER-2 extracellular domain. The vaccine was administered alone, thanks to the intrinsic adjuvanticity of the VLP, or with Montanide ISA 51. QD is a HER-2-positive cell line established from a mammary carcinoma of a Delta16 (FVB background) transgenic mouse, bearing the human HER-2 splice variant Delta16. FVB female mice were challenged in the mammary fat pad with 1 million QD cells; vaccinations started 2 weeks after cell challenge and were repeated every 2 weeks. Untreated mice developed progressive tumors within one month, whereas 70% of mice vaccinated without adjuvant and all mice vaccinated with adjuvant were still tumor-free one year after cell challenge. Mice challenged intravenously (i.v.) developed more than 300 lung metastases, whereas all vaccinated mice remained metastasis-free. Delta16 transgenic mice are immunologically tolerant to human HER-2 and develop aggressive mammary carcinomas with a median latency of 5 months. Vaccination of young, tumor-free Delta16 mice completely prevented tumor onset for more than one year. Delta16 mice challenged i.v. with QD cells mice developed a mean of 68 lung nodules, whereas 73% of mice therapeutically vaccinated without adjuvant, and all mice vaccinated with E2SB-C001+ISA 51, were free from metastases. ES2B-C001 induced copious anti-HER-2 antibodies of all IgG subclasses, ranging 1-10 mg/mL in FVB mice and 0.1-1 mg/mL in Delta16 mice; antibody titers remained very high for 6-10 months after the last vaccination. Antibodies inhibited the 3D growth of human HER-2+++ and HER-2++ breast cancer cells, of trastuzumab resistant cells and of gastric carcinoma cells. Vaccination increased interferon-gamma secreting cells in the spleen, as evaluated by ELISPot (21±3 spots/2x105 cells). The results warrant further development of ES2B-C001 for the therapy of HER-2 positive human breast cancer and of other tumors expressing HER-2. Citation Format: Francesca Ruzzi, Arianna Palladini, Stine Clemmensen, Annette Strobaek, Nicolaas Buijs, Tanja Domeyer, Jerzy Dorosz, Vladislav Soroka, Dagmara Grzadziela, Christina Jo Rasmussen, Ida Busch Nielsen, Max Soegaard, Maria Sofia Semprini, Laura Scalambra, Stefania Angelicola, Lorena Landuzzi, Pier-Luigi Lollini, Mette Thorn. Preclinical activity of ES2B-C001, a human candidate HER-2 virus-like particle (VLP) vaccine, against mammary carcinoma onset and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 687.

In the presence of non-neutralising maternally derived antibodies, intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells

The purpose of this study was to compare the immune response generated by the intramuscular and the intradermal vaccination route against the porcine reproductive and respiratory syndrome virus (PRRSV). Piglets from a seronegative and a seropositive farm were selected (n = 28 piglets per farm), and each group was divided into two groups and vaccinated after weaning with modified live vaccine Unistrain® PRRS (Laboratorios Hipra Amer, Spain) by the intramuscular (IM) or the intradermic (ID) route. For the following 6 weeks, animals were weekly bled to assess the humoral response by PRRSV-specific antibody ELISA and viral neutralisation test. At 0-, 3-, 4- and 6 weeks post-vaccination, peripheral mononuclear blood cells (PBMC) from eight animals per group were recovered to analyse cellular response by IFN-γ ELISPOT and lymphoproliferation. Serum IL-12 was also quantified by ELISA. Seroconversion was first detected 14 days post-vaccination (dpv) for both IM and ID routes, and peaked at 35 dpv (both IM groups and ID seropositive) or 42 dpv (ID seronegative). At 3 weeks after vaccination, 6/27 (22.22%) animals from negative origin had not seroconverted, and neutralising titres were significantly lower at 35 dpv compared to the seropositive origin (mean log2 titres of 1.36 and 4.25 respectively) Also, it was 10 times more probable for them to have high levels of IL-12 a week after vaccination than for animals of seropositive origin. Cellular immune response analysed by lymphoproliferation and IFN-γ ELISPOT was already present at 21 dpv and until 42 dpv, with no significant differences between groups except for a higher lymphoproliferation at 35 dpv in the IM seropositive group (Kruskal-Wallis, p < 0.05). These results indicate that the intradermal route induces an immune response equivalent to the classical intramuscular route even in presence of non-neutralising maternal immunity, which in this study has proven to facilitate seroconversion after vaccination with an heterologous strain.

Immune response does not prevent homologous Porcine epidemic diarrhoea virus reinfection five months after the initial challenge.

The aim of the present study was to evaluate the duration of protective immunity against Porcine epidemic diarrhoea virus (PEDV). To do so, a two phases study was performed. In the first phase, 75 four-week-old pigs (group A) were orally inoculated (0days post-inoculation; dpi) with a European PEDV G1b strain and 14 were kept as controls (group B). The second phase started five months later (154 dpi), when animals in group A were homologous challenged and animals in group B were challenged for first time. Clinical signs, viral shedding and immune responses were evaluated after each inoculation, including the determination of antibodies (ELISA and viral neutralization test, IgA and IgG ELISPOTs using peripheral blood mononuclear cells and lymph node cells) and the frequency of interferon-gamma (IFN-γ) secreting cells. During the first phase, loose stools/liquid faeces were observed in all group A animals. Faecal shedding of PEDV occurred mostly during the first 14days but, in some animals, persisted until 42 dpi. All inoculated animals seroconverted for specific-PEDV IgG and IgA, and for neutralizing antibodies (NA). At 154 dpi, 77% of pigs were still positive for NA. After that, the homologous challenge resulted in a booster for IgG, IgA, NA, as well as specific-PEDV IgG, IgA and IFN-γ secreting cells. In spite of that, PEDV was detected in faeces of all pigs from group A, indicating that the immune response did not prevent reinfection, although the duration of the viral shedding and the total load of virus shed were significantly lower for previously challenged pigs (p<.05). Taken together, the results indicated that, potentially, maintenance of PEDV infection within an endemic farm may occur by transmission to and from previously infected animals and also indicates that sterilizing immunity is shorter than the productive life of pigs.

Design and characterization of a consensus hemagglutinin vaccine immunogen against H3 influenza A viruses of swine

The substantial genetic diversity exhibited by influenza A viruses of swine (IAV-S) represents the main challenge for the development of a broadly protective vaccine against this important pathogen. The consensus vaccine immunogen has proven an effective vaccinology approach to overcome the extraordinary genetic diversity of RNA viruses. In this project, we sought to determine if a consensus IAV-S hemagglutinin (HA) immunogen would elicit broadly protective immunity in pigs. To address this question, a consensus HA gene (designated H3-CON.1) was generated from a set of 1,112 H3 sequences of IAV-S recorded in GenBank from 2011 to 2015. The consensus HA gene and a HA gene of a naturally occurring H3N2 IAV-S strain (designated H3-TX98) were expressed using the baculovirus expression system and emulsified in an oil-in-water adjuvant to be used for vaccination. Pigs vaccinated with H3-CON.1 immunogen elicited broader levels of cross-reactive neutralizing antibodies and interferon gamma secreting cells than those vaccinated with H3-TX98 immunogen. After challenge infection with a fully infectious H3N2 IAV-S isolate, the H3-CON.1-vaccinated pigs shed significantly lower levels of virus in their nasal secretions than the H3-TX98-vaccinated pigs. Collectively, our data provide a proof-of-evidence that the consensus immunogen approach may be effectively employed to develop a broadly protective vaccine against IAV-S.

Differences in monocyte: lymphocyte ratio and Tuberculosis disease progression in genetically distinct populations of macaques

Monocyte:lymphocyte ratio (M:L) has been identified as a risk factor in development of TB disease in children and those undergoing treatment for HIV in co-infected individuals. Retrospective analysis was performed using M:L data collected from TB modelling studies performed in Rhesus macaques of Indian genotype (RM), cynomolgus macaque of Chinese genotype (CCM) and cynomolgus macaque of Mauritian genotype (MCM), which found that the more susceptible populations (RM and MCM) had higher M:L ratios than the least susceptible population (CCM). Following Mycobacterium tuberculosis exposure, significant increases in M:L ratio were observed in susceptible RM and MCM within 12 weeks of TB infection, whereas M:L in CCM remained stable, suggesting that changes in M:L ratio may also act as a biomarker of TB disease progression. The frequency of PPD-specific interferon gamma (IFNγ) secreting cells (SFU) were compared, with the more susceptible macaque populations showing an association between M:L and IFNγ SFU frequency. Investigation of the genes associated with monocyte-derived antigen presenting cells revealed differences between RM and CCM, highlighting differences in their monocyte populations, as well as overall M:L ratio. Differences in M:L ratio between macaque populations could be used to explore immunological mechanisms in susceptible populations that would complement human population studies.

Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes.

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.

Specific antitumour immunity of HIFU-activated cytotoxic T lymphocytes after adoptive transfusion in tumour-bearing mice

Purpose: The aim of this study was to investigate the specific anti-tumour immunity of cytotoxic T lymphocytes (CTL) activated by high-intensity focused ultrasound (HIFU) after adoptive transfer in a murine tumour model. Materials and methods: H22 tumour-bearing mice were treated by either HIFU or sham-HIFU, while naïve syngeneic mice were used as controls. They were sacrificed and the spleens were harvested 14 days after HIFU. T lymphocytes were obtained from the spleens, and then adoptively transferred into 40 mice each bearing a 3-day implanted H22 tumour. On day 14 after adoptive transfer, 10 mice were sacrificed in each group for assessment of the number of tumour-infiltrating T lymphocytes and interferon-gamma (IFN-γ) secreting cells. The remaining 30 mice were continuously observed for 60 days, and tumour growth, progression and survival were recorded. Results: HIFU significantly increased peripheral blood CD3+, CD4+ levels and CD4+/CD8+ ratio (P < 0.05), CTL cytotoxicity (P < 0.01) and IFN-γ and TNF-α secretion (P < 0.01) in H22 tumour-bearing mice. Adoptive transfer of HIFU-activated T lymphocytes into the autologous tumour-bearing mice induced a significant increase of tumour-infiltrating T lymphocytes and IFN-γ-secreting cells (P < 0.001). Compared to the control and sham-HIFU groups, HIFU-activated lymphocytes elicited significant inhibition of in vivo tumour growth (P < 0.01) and progression (P < 0.0001), and longer survival time in the tumour-bearing mice (P < 0.001). Conclusions: HIFU could enhance CTL’s specific antitumour immunity. Adoptive transfer of HIFU-activated T lymphocytes could increase local antitumour immunity, and elicit stronger inhibition on tumour growth and progression, with more survival benefit in the autologous tumour-bearing mice.

Comparison of Two Commercial Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Modified Live Vaccines against Heterologous Type 1 and Type 2 PRRSV Challenge in Growing Pigs.

The objective of the present study was to compare the efficacy of two commercial type 1 porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccines against heterologous type 1 and type 2 PRRSV challenge in growing pigs. Vaccination with a type 1 PRRSV vaccine reduced the level of viremia after type 1 PRRSV challenge but did not reduce the level of viremia after the type 2 PRRSV challenge in pigs. Increased levels of interleukin-10 (IL-10) stimulated by type 2 PRRSV coincided with the low numbers of type 2 PRRSV-specific interferon gamma-secreting cells (IFN-γ-SC) in vaccinated pigs after type 2 PRRSV challenge, whereas low levels of IL-10 stimulated by type 1 PRRSV coincided with high numbers of type 1 PRRSV-specific IFN-γ-SC in vaccinated pigs after type 1 PRRSV challenge. Additionally, vaccination with the type 1 PRRSV vaccine effectively reduced the lung lesions and type 1 PRRSV nucleic acids in type 1 PRRSV-challenged pigs but did not reduce lung lesions and type 2 PRRSV nucleic acids in type 2 PRRSV-challenged pigs. There were no significant differences between two commercial type 1 PRRSV vaccines against type 1 and type 2 PRRSV challenge based on virological results, immunological responses, and pathological outcomes. This study demonstrates that vaccinating pigs with the type 1 PRRSV vaccine provides partial protection against respiratory disease with heterologous type 1 PRRSV challenge but no protection with heterologous type 2 PRRSV challenge.

Comparison of Four Commercial One-Dose Porcine Circovirus Type 2 (PCV2) Vaccines Administered to Pigs Challenged with PCV2 and Porcine Reproductive and Respiratory Syndrome Virus at 17 Weeks Postvaccination To Control Porcine Respiratory Disease Complex under Korean Field Conditions

Under Korean field conditions, coinfection with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is most commonly observed in porcine respiratory disease complex (PRDC). Despite the wide use of PCV2 vaccination, PRDC remains a serious respiratory problem. Thus, the objective of this study was to determine and compare the efficacy of 4 one-dose PCV2 vaccines on 3-week-old pigs with an experimental PCV2-PRRSV challenge at 17 weeks postvaccination. Regardless of which commercial PCV2 vaccine was used, the vaccination of piglets at 3 weeks of age was efficacious against cochallenge of PCV2 and PRRSV, on the basis of growth performance and PCV2-associated lesions. However, the inactivated chimeric PCV1-2 and the PCV2 vaccines induced higher PCV2-specific neutralizing antibody (NA) titers and PCV2-specific gamma interferon-secreting cells and lower PCV2 viremia levels than the two PCV2 subunit vaccines. The vaccination of piglets against PCV2 at 3 weeks of age was effective in reducing PCV2 viremia and PCV2-associated lesions during the finishing period, which is an age at which pigs are frequently affected by PRDC caused by coinfection with PCV2 and PRRSV under Korean field conditions.

Comparative Effects of Vaccination against Porcine Circovirus Type 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in a PCV2-PRRSV Challenge Model

The objective of the present study was to determine the effects of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) vaccinations in an experimental PCV2-PRRSV challenge model, based on virological (viremia), immunological (neutralizing antibodies [NAs], gamma interferon-secreting cells [IFN-γ-SCs], and CD4(+) CD8(+) double-positive cells), and pathological (lesions and antigens in lymph nodes and lungs) evaluations. A total of 72 pigs were randomly divided into 9 groups (8 pigs per group): 5 vaccinated and challenged groups, 3 nonvaccinated and challenged groups, and a negative-control group. Vaccination against PCV2 induced immunological responses (NAs and PCV2-specific IFN-γ-SCs) and reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PCV2 did not affect the PRRSV immunological responses (NAs and PRRSV-specific IFN-γ-SCs), PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. Vaccination against PRRSV did not induce immunological responses (PRRSV-specific IFN-γ-SCs) or reduce PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. In addition, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. In summary, vaccination against PCV2 reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. Therefore, the PCV2 vaccine decreased the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs. In contrast, the PRRSV vaccine alone did not decrease the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs.

Risk of Immunodeficiency Virus Infection May Increase with Vaccine-Induced Immune Response

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).

Protective effect of the maternally derived porcine circovirus type 2 (PCV2)-specific cellular immune response in piglets by dam vaccination against PCV2 challenge

The objective of the present study was to evaluate (i) the passive transfer of maternally derived functional porcine circovirus type 2 (PCV2)-specific lymphocytes of seronegative sows immunized with the PCV2 vaccine to newborn piglets and (ii) the functional role of the maternally derived PCV2-specific cellular immune response in protecting newborn piglets from challenge with PCV2. After ingesting colostrums, piglets from vaccinated sows (PT01 and PT02) have significantly higher numbers of PCV2-specific gamma interferon-secreting cells, an increased PCV2-specific delayed type hypersensitivity response, and a stronger proliferative response of peripheral blood mononuclear cells compared with piglets from non-vaccinated seronegative sows (PT03 and PT04). In the PCV2 challenge study, the number of serum genomic PCV2 copies was significantly less in piglets from vaccinated sows (PT02) compared with piglets from non-vaccinated sows (PT04) at 7-28 days post-inoculation (P<0.05 and P<0.001). The histopathological lesions and immunohistochemical scores were significantly lower in piglets of vaccinated sows compared with those of non-vaccinated sows. To our knowledge, this is the first report of transferring a maternally derived PCV2-specific cellular immune response from vaccinated dams to their offspring. Maternally derived adaptive cellular immune responses play a critical role in protecting newborn piglets challenged with PCV2 at 3 weeks of age.

CMV-Reactive T Cells Can Be Isolated From G-CSF Mobilised Peripheral Blood Apheresates.

AbstractAbstract 1276Reactivation of cytomegalovirus (CMV) post allogeneic stem cell transplant (SCT) has shown to be controlled by the adoptive transfer of donor derived CMV-reactive T cells (CMV-T). A current method for the generation of CMV-T cells is CMV antigenic stimulation of donor peripheral blood mononuclear cells (PBMC), followed by isolation of interferon-gamma (IFN-g) secreting cells, by magnetic enrichment. The feasibility of generating a CMV-T cell product for clinical use, using PBMC from a G-CSF mobilised donor stem cell apheresis has yet to be fully determined.Small aliquots (<1ml) were taken from the mobilised apheresis of CMV positive donors (n=5), the PBMC separated using density gradient centrifugation and then stimulated in a 16 hour culture using a commercially available CMVpp65 peptide pool (Peptivator – Miltenyi Biotec). CMV-T cells were then isolated using both IFN-g secretion and expression of the activation marker CD25. Results were then compared directly against the same methods in non-mobilised PBMC. [Display omitted] The proportion of CMVpp65 stimulated CD25+ T cells was equivalent between mobilised (53.15%) and non-mobilised (63.28%) PBMC. The CMV-T response was also predominantly CD4+ in both mobilised (98%) and non-mobilised (88%) PBMC. In contrast IFN-g secretion was shown to identify CMV-T in mobilised PBMC (41.77%), but this was consistently 50–60% lower in frequency compared to non-mobilised PBMC (95.59%). This 1–2 fold reduction in IFN-g+ T cell activation does not appear to effect the ratio of CD4+ to CD8+ T cells, which was comparable between mobilised (1:1.1) and non-mobilised (1:1.5) PBMC (Figure 1).Furthermore a reduction was observed in both yield (6.62% vs. 58.93%) and purity (35.68 vs. 76.01%) post enrichment of IFN-g+ CMV-T cells from mobilised PBMC. Of the IFN-g+ cells isolated from non-mobilised PBMC ∼30% were also positive for CD25 whereas this number was undetectable (<1%) in the mobilised setting) (Figure 2a and b). [Display omitted] [Display omitted] Enrichment of CD25+ CMV-T cells showed parity between mobilised and non-mobilised PBMC in terms of both yield (26.12% vs. 34.75%) and purity (89.92% vs. 93.39%). We have also observed that enrichment of CD25+ CMV-T cells depleted the naturally occurring CD25+ T cells seen in the unstimulated PBMC. Background expression of CD25 has been thought previously to be a barrier to selection of activated cells by CD25. However, CD25+ selections from non-stimulated cultures resulted in a yield of <1%.These results indicate that G-CSF can suppress the Ag-specific T cell responses following in vitro stimulation with a CMVpp65 peptide pool but CMV-T can still be detected and isolated. We hypothesise that the reduced IFN-g+ T cell activation in mobilised PBMC could be as a result of the suppressive nature of an increased frequency of FoxP3+ IL-10 secreting T cells compared to that in non-mobilised PBMC.We have demonstrated G-CSF suppression of CMVpp65 stimulated T cell responses with reference to a type 1 cytokine profile. Both CD25+ and IFN-g+ CMV-T from mobilised and non-mobilised PBMC are being tested for secretion of a profile of type 1 and type 2 cytokines to include TNF-α, IL-2, IL-4 and IL-10. Direct selection of CMV-T by CD25 expression represents a potential approach for isolating these cells for clinical use.ES, KN and this work were supported by a grant from the Technology Strategy Board (TSB) of the UK Government Department of Business, Innovation & Skills Disclosures:No relevant conflicts of interest to declare.

Rapid Generation of Full Clinical-Grade Human Antiadenovirus Cytotoxic T Cells for Adoptive Immunotherapy

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

Crucial Role of Interferon-γ in Experimental Autoimmune Prostatitis

An autoimmune etiology is proposed in some patients with chronic nonbacterial prostatitis since they show interferon-gamma secreting lymphocytes specific to prostate antigens in the periphery and increased interferon-gamma in seminal plasma. We investigated the involvement of interferon-gamma in an animal model of autoimmune prostatitis. Experimental autoimmune prostatitis was studied in the no-obese diabetic and C57Bl/6 (Harlan, Zeist, The Netherlands) susceptible mouse strains, and in the IRF-1 KO and STAT-1 KO mouse strains deficient in transcription factors involved in interferon-gamma signaling. Experimental autoimmune prostatitis was characterized by prostate specific interferon-gamma secreting cells in the periphery and by T-helper 1 related cytokines in the target organ. Increased interferon-gamma and interleukin-12 were observed in the prostate of autoimmune animals while interleukin-10 and interleukin-4 were decreased and unaltered, respectively. The absence of transcription factors involved in the interferon-gamma signaling cascade, IRF-1 and STAT-1, made mice resistant to experimental autoimmune prostatitis. IRF-1 KO and STAT-1 KO mice immunized with prostate antigens did not show infiltration or alterations in the prostate. They did not have the typical prostate specific autoimmune response and showed decreased interferon-gamma, interleukin-12 and interleukin-10, and augmented interleukin-4 in the prostate. Our results argue for a crucial role of interferon-gamma as a key factor in the pathogenesis of the disease. Intense research is promptly required to identify the pathogenic mechanisms underlying chronic prostatitis/chronic pelvic pain syndrome to find a more rational therapy.

Gemini nanoparticles as a co-delivery system for antigen &amp;ndash; CpG oligodeoxynucleotide adjuvant combination

Enhancement of antigen-specific immune responses could be achieved by co-delivery of antigens and adjuvants. CpG oligodeoxynucleotides (unmethylated cytosine-guanine tandems in a specific sequence; ODNs) increase innate and antigen-specific immune responses. Effective co-delivery approaches of CpG ODNs and antigens to antigen-presenting cells are needed to achieve more potent antigen-specific immune responses. We evaluated both cellular and humoural immune responses triggered by hen egg lysozyme (HEL), a model antigen, and CpG ODNs formulated in gemini surfactant and dioleoyl phosphatidylcholine-based nanoparticles as an intradermal and topical co-delivery system in a murine animal model. Overall, intradermal injection of HEL/CpG nanoparticles induced a more pronounced Th1 immune response compared with the HEL and HEL/CpG topical formulations, as evidenced by the shift in the Th2 response triggered by the antigen alone to a mixed Th1/Th2 immune response and increased the presence of interferon-gamma (IFN-γ) secreting cells in the spleen. However, in case of topical administration, the nanoparticle formulation of HEL produced enhanced immune response and immunomodulation even without the incorporation of CpG. The HEL-specific immune response and Th1 bias demonstrated the advantage of co-delivery of HEL/CpG ODNs by gemini nanoparticles intradermally, whereas, the adjuvant effect of the nanoparticle delivery system itself was more significant after topical treatment.

Adoptive Transfer of CMV-Specific Donor T Cells Following Allogeneic Transplantation Leads to Rapid and Safe Systemic Reconstitution.

Abstract 44Reactivation of CMV remains a significant problem following allogeneic hematopoietic stem cell transplantation. Antiviral drug therapy is effective but toxic, and resistant strains of CMV are increasingly being reported. Virus-specific T lymphocytes are necessary for the control of viral reactivation. Adoptive transfer of donor derived CMV-specific T cells has been reported previously but most methods to produce such cells have involved several weeks of in vitro culture or have produced a therapeutic product restricted to CD8 T cells. The current method involves a short incubation of donor peripheral blood mononuclear cells with either CMV-pp65 protein (20 hours) or a pool of peptides from pp65 (6 hours) with subsequent isolation of interferon-gamma secreting cells by CliniMACS using IFNψ capture microbeads (Miltenyi Biotec). This technique permits rapid isolation of an enriched IFNψ secreting T cell product, manufactured to clinical grade, which is then cryopreserved in dosed aliquots for subsequent infusion. Here we report the outcome of a single arm phase I/II in which CMV-T cells given pre-emptively at first detection (qPCR) of CMV DNA in peripheral blood, or at day +40-60 as prophylaxis. CMV replication was monitored by weekly PCR and reconstitution of CMV-specific T cells by pentamer labelling and/or IFNψ secretion assay. Conventional antiviral drug therapy was instituted if the viral load rose above institutional threshold. 30 recipients of T cell depleted low intensity transplants from HLA-matched CMV-seropositive related donors were enrolled between 2006 and 2008. Donors underwent a second, short apheresis procedure approximately 15 days after collection of the mobilised HPC-A for the collection of CMV-T cells. 26 clinical-grade products were produced to full cGMP standards; four donors were unsuitable or withdrew. The mean yield of cells following enrichment was 41.7% with a median purity of 43.9% (range 1.4-81.8). Adequate CMV-T cells were isolated from all donors. Both pp65 and peptide stimulated products contained both CD4 and CD8 reactive T cells. Median dose of CMV-specific CD4 T cells was 2840/kg and of CMV-specific CD8 was 630/kg. Eighteen patients received a single dose of 1×10^4 CD3+/kg; 13 were CMV seropositive; 11 were treated pre-emptively and 7 prophylactically. 83% had received T cell deplete regimens. Within 2 weeks of infusion in vivo expansion of CMV-T cells was observed in 17 of 18 patients. One patient required 4 weeks to generate detectable CMV-T cell in his peripheral blood. TCR-BV usage of the CMV-T cells post infusion matched that of the cells which had been infused. The 7 patients who had cells infused prophylactically all showed expansions of CMV-T cells in the absence of detectable viral DNA in peripheral blood. Subsequent low level CMV-reactivation was seen in one of these and was associated with rapid CMV-T cell expansion with clearance of virus without anti-viral drug therapy. One developed subsequent extensive chronic GvHD and required antiviral treatment for multiple reactivation episodes following introduction of steroids. Of the 11 patients treated pre-emptively, 9 received antiviral therapy for the initial reactivation, although in 7 patients this was required for only 7-15 days. (compared to a median of 21 days in historical controls). Three patients had a further CMV reactivation event. One followed prednisolone therapy for acute grade II GvHD. The second was the patient who had shown poor T cell expansion post infusion and had required prolonged anti-viral therapy (33 days) for the initial CMV reactivation. The third patient received no treatment and cleared virus following a further in vivo expansion of CMV-reactive T cells, suggesting the presence of a functional memory population. GVHD incidence and severity was no worse than seen in comparable historical controls. 3 patients suffered grade 2-3 acute GvHD. 3/17 evaluable patients developed extensive chronic GvHD (2 were recipients of T replete grafts). 16/18 patients were alive at the end of the 6 month monitoring period and CMV-reactive T cells were detectable in all 16. CMV-specific donor T cells can be readily produced to cGMP compliance which can be safely infused and lead to early immune reconstitution in at-risk patients. Cells expand in response to subsequent CMV-reactivation and patients appear to require fewer anti-viral treatment episodes which is being tested in an ongoing phase III trial. Disclosures:Lowdell:Cell Medica Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.

Susceptibility to Simian Immunodeficiency Virus Ex Vivo Predicts Outcome of a Prime-Boost Vaccine After SIVmac239 Challenge

Efficacy assessment of AIDS vaccines relies both on preclinically challenging immunized monkeys with simian immunodeficiency virus (SIV) or monitoring infection rates in large human trials. Although conventional parameters of vaccine-induced immune responses do not completely predict outcome, existing methods for testing cellular immunity are sophisticated and difficult to establish in resource-limited settings. We have used virus replication kinetics (VVR) on ConA-stimulated peripheral blood mononuclear cells from rhesus monkeys immunized with DNA replication-defective adenovirus vector expressing various SIV genes, as an ex vivo model, to mimic the effects of different immune effector functions on viral infection. VVR was attenuated by the immunization and correlated 2 weeks after first boost, with the number of interferon gamma-secreting cells and T-cell noncytotoxic antiviral responses. Importantly, VVR on the day of challenge but not interferon gamma responses correlated with viremia and with memory CD4+ T-cell measurements after SIVmac239 challenge. Similarly, T-cell noncytotoxic antiviral responses on the day of challenge correlated directly with memory CD4 T cell and inversely with plasma viremia after challenge. VVR thus served as a better predictor of protective capacity of the vaccine regimen in these monkeys. We suggest that VVR be considered in the evaluation of candidate AIDS vaccines in humans.

Elastase-Dependent Live Attenuated Swine Influenza A Viruses Are Immunogenic and Confer Protection against Swine Influenza A Virus Infection in Pigs

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.