Cell-mediated immune responses to inactivated trivalent influenza-vaccination are decreased in patients with common variable immunodeficiency.

Influenza-specific cell-mediated immune (CMI) responses can protect from influenza, but may be decreased in CVID-patients since defects in CMI responses have been demonstrated in CVID-patients. Therefore CMI responses were evaluated in 15 CVID-patients and 15 matched healthy controls (HC) by determining frequencies of interferon (IFN)γ-producing PBMC, and frequencies of IFNγ-, interleukin (IL)-2- and tumour necrosis factor (TNF)α-producing CD4+ and CD8+ T-cells before and after influenza vaccination using IFNγ enzyme-linked immunospot (IFNγ-ELISpot) and flow cytometry. Humoral responses were determined using haemagglutination inhibition assay. In CVID-patients the number of spotforming PBMC in the IFNγ-ELISpot did not increase following influenza vaccination, in contrast to HC. In flow cytometry, the frequencies of IFNγ-producing T-cells decreased in CVID-patients after influenza vaccination, while in HC the frequencies of IFNγ-production flow cytometry increased. Concluding, CMI responses following influenza vaccination are hampered in CVID-patients compared to HC. Additional protective strategies against influenza other than vaccination are warranted.

Patients with humoral primary immunodeficiency do not develop protective anti-influenza antibody titers after vaccination with trivalent subunit influenza vaccine.

Yearly influenza vaccination is recommended for patients with humoral primary immunodeficiency (hPID). However, humoral responses following vaccination can be expected to be reduced in these patients. The efficacy of influenza vaccination in patients with hPID, anti-influenza antibody responses was assessed in 26 patients with hPID and 26 matched healthy controls (HC) using hemagglutination inhibition assay. Following vaccination, geometric mean titers (GMT) significantly increased for all influenza strains in the HC group, but only for A/H1N1 in the patient group. Fold increase in anti-influenza titer and seroprotection rates were lower for patients than for HC for A/H3N2 and A/H1N1, leading to postvaccination titer > or =40 in only 29% and 83% vs. 77% and 100%, respectively. Previous vaccination in patients and treatment with IVIg did not result in a higher rate of postvaccination titer > or =40. In conclusion, patients with hPID show hardly any humoral response following influenza vaccination.

Cell-mediated immune responses to influenza vaccination in Wegener's granulomatosis.

BACKGROUND: Both antibody and cell-mediated immune responses are involved in the defence against influenza. In Wegener's granulomatosis (WG), antibody responses to influenza vaccination appear to be similar to those in healthy controls, but cell-mediated responses have not been studied. OBJECTIVE: To determine whether cell-mediated responses to influenza vaccination in WG vary from those in controls. METHODS: Twenty-five patients with WG and healthy controls received subunit influenza vaccine. Peripheral blood mononuclear cells were obtained before and 1 month after vaccination. Cell-mediated responses to A/H1N1 and A/H3N2 were assessed using interferon gamma (IFN gamma) ELISpot and intracellular cytokine staining for IFN gamma, tumour necrosis factor and interleukin 2. RESULTS: Before vaccination, patients and controls showed similar recall responses to A/H1N1 and A/H3N2. After vaccination, patients and controls showed similar levels of increase in spot-forming cells against A/H1N1 and A/H3N2. By flow cytometry, upon vaccination, proportions of cytokine-producing CD4 T cells increased in patients and controls for A/H1N1 and A/H3N2. CONCLUSIONS: Cell-mediated responses to influenza vaccination in patients with WG are comparable to those in healthy controls.

Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling.

In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.

Effect of a second, booster, influenza vaccination on antibody responses in quiescent systemic lupus erythematosus: an open, prospective, controlled study.

OBJECTIVE: In SLE, a decreased antibody response on influenza vaccination has been reported. In this study, we assessed whether a booster vaccination could improve antibody responses, as determined by seroprotection rates, in SLE patients. METHODS: SLE patients (n = 52) with quiescent disease (SLEDAI < or =4) and healthy controls (HCs) (n = 28) received subunit influenza vaccine in October-December 2007. After 4 weeks, only SLE patients received a second dose of vaccination. Sera were obtained before both vaccinations, and 4 weeks after the second vaccination. At each visit, SLE disease activity was recorded. The haemagglutination inhibition test was used to measure antibody titres. Seroprotection was defined as a titre > or =40. RESULTS: Following the first vaccination, seroprotection rates and geometric mean titres (GMTs) to each vaccine strain increased in both SLE patients and controls to comparable levels. Seroprotection rates in SLE patients after the first vaccination were 86.5% to A/H1N1, 80.8% to A/H3N2 and 61.5% to the B-strain while GMTs were 92.6, 56.2 and 39.2, respectively. Overall, the booster vaccination did not lead to a further rise of seroprotection rates and GMTs in SLE patients. However, in patients not vaccinated in the previous year, GMT and seroconversion rate to A/H1N1 did rise following the booster vaccination. Both influenza vaccinations did not increase SLEDAI scores. CONCLUSIONS: Additional value of a booster influenza vaccination in SLE is limited to patients who were not vaccinated in the previous year.

Monophosphoryl Lipid A-Adjuvanted Virosomes with Ni-Chelating Lipids for Attachment of Conserved Viral Proteins as Cross-Protective Influenza Vaccine.

Induction of CD8+ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross-protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen-presenting cells (APCs) and get access to the MHC class I antigen processing pathway. To facilitate the generation of NP-specific CTL immunity the authors develop a novel influenza vaccine consisting of virosomes with the Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal-ion-chelating lipid DOGS-NTA-Ni incorporated in the membrane. In vitro, virosomes with incorporated MPLA induce stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS-NTA-Ni show high conjugation efficacy for his-tagged proteins and facilitate efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA-adjuvanted virosomes with attached NP results in priming of NP-specific CTLs while MPLA-adjuvanted virosomes with admixed NP are inefficient in priming CTLs. Both vaccines induce equally high titers of NP-specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP are protected from severe weight loss. Yet, unexpectedly, they show more weight loss and more severe disease symptoms than mice immunized with MPLA-virosomes without NP. Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen-specific antibody responses in vivo. However, for protection from influenza infection NP-specific immunity appears not to be advantageous.

Porcine fetal ventral mesencephalic cells are targets for primed xenoreactive human T cells.

Xenotransplantation of porcine fetal ventral mesencephalic (pfVM) cells to overcome the dopamine shortage in the striatum of patients with Parkinson's disease seems a viable alternative to allotransplantion of human fetal donor tissue, especially because the latter is complicated by both practical and ethical issues. There is, however, little known about the xenospecific immune responses involved in such an intracerebral xenotransplantation. The aim of our study was to investigate whether (1) naive human peripheral blood mononuclear cells (PMBC) display cytotoxicity against pfVM cells of E28 pig fetuses, and (2) priming of human PBMC by xenogeneic antigen presenting cells (APC) modulates pfVM-directed cellular cytotoxicity. For this purpose fresh PMBC from nine individual donors were primed by incubation with either irradiated pfVM cells or porcine spleen cells (PSC) as APC in the presence of IL-2 for 1 week before assessing cytotoxicity in a 51Cr release assay. Also, direct NK reactivity and antibody-dependent cellular cytotoxicity (ADCC) of fresh PMBC against pfVM cells was assessed. No direct cytotoxicity of naive cells (either NK reactivity or ADCC) against pfVM cells could be determined. Only PMBC primed with PSC were capable of lysing pfVM cells. PBMC primed with pfVM cells did not show cytolytic capacity towards pfVM. Interestingly, large differences in xenospecific T-cell responses exist between individual donor PBMC. Thus, human T cells are capable of killing pfVM cells in a xenoreactive response, but only after priming by donor APC. The large interindividual differences between human donors in their xenoreactive response may influence patient selection for xenotransplantation and chances of graft survival for individual patients.

The modulation of angiogenesis in the foreign body response by the poxviral protein M-T7.

The foreign body response is characterized by enhanced recruitment of inflammatory cells. As the directional movement of cells is controlled by chemokines, disruption of the chemokine network would be an attractive approach to improve biocompatibility of an implanted material. The sequestration of chemokines by cell surface-expressed glycosaminoglycans (GAGs) is vital for in vivo chemokine activity. The myxoma virus encodes a soluble protein, M-T7, that interacts with conserved GAG-binding domains of chemokines to block chemokine-mediated leukocyte recruitment. We hypothesized that M-T7 might also affect the function of other inflammation-associated proteins in addition to chemokines that bind to GAG. In our studies, we focussed on the modulation of the GAG-binding molecules macrophage chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor-164 (VEGF164) in the inflammatory reaction against subcutaneously implanted degradable cross-linked dermal sheep collagen discs in AO rats. Genetic delivery of M-T7 delays the influx of macrophages into the collagen discs. In addition, angiogenesis around the implanted material was reduced. The discs revealed reduced levels of rat MCP-1 and rat VEGF164. This was not due to down regulation of transcription of the genes that encode MCP-1 and VEGF164. Our in vivo observations suggest that, in addition to chemokines such as MCP-1, M-T7 neutralizes VEGF164.

Integrity of airway epithelium is essential against obliterative airway disease in transplanted rat tracheas.

BACKGROUND: The pathogenesis of obliterative bronchiolitis after lung transplantation requires further elucidation. In this study we used rat trachea transplantation to examine the role of epithelium in the progression of obliterative airway disease. METHODS: Normal and denuded (i.e., epithelium removed) trachea grafts from Lewis (LEW) and Brown Norway (BN) rats were transplanted sub-cutaneously into LEW rats. Viable trachea epithelial cells (to recover epithelium) were seeded into the lumen of some of the denuded tracheas. Grafts were removed at different time-points between 2 days and 8 weeks after transplantation. Histologic analysis was performed to evaluate cellular infiltration of inflammatory cells, loss of epithelium, and obliteration of trachea lumen. RESULTS: Obliteration was found to occur in trachea transplants after loss of epithelium, caused by rejection in allografts or by enzymatic denudation in isografts. In these situations, fibroblasts started to proliferate and migrate into the lumen in the second week after transplantation. Obliteration could be prevented when epithelial integrity was restored by seeding epithelial cells; no obliteration occurred when denuded trachea isografts were seeded with epithelial cells, whereas non-seeded denuded tracheas were obliterated at Day 6 after transplantation. CONCLUSIONS: We conclude that integrity of airway epithelium is essential for rat trachea transplants to be safeguarded from obliterative airway disease. For clinical lung transplantation the results of our study suggest that protection of the integrity of airway epithelium may be important in preventing the development of obliterative bronchiolitis.

Comparison of adjuvants for a spray freeze-dried whole inactivated virus influenza vaccine for pulmonary administration.

Stable vaccines administered to the lungs by inhalation could circumvent many of the problems associated with current immunizations against respiratory infections. We earlier provided proof of concept in mice that pulmonary delivered whole inactivated virus (WIV) influenza vaccine formulated as a stable dry powder effectively elicits influenza-specific antibodies in lung and serum. Yet, mucosal IgA, considered particularly important for protection at the site of virus entry, was poorly induced. Here we investigate the suitability of various Toll-like receptor (TLR) ligands and the saponin-derived compound GPI-0100 to serve as adjuvant for influenza vaccine administered to the lungs as dry powder. The TLR ligands palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN) as well as GPI-0100 tolerated the process of spray freeze-drying well. While Pam3CSK4 had no effect on systemic antibody titers, all the other adjuvants significantly increased influenza-specific serum and lung IgG titers. Yet, only GPI-0100 also enhanced mucosal IgA titers. Moreover, only GPI-0100-adjuvanted WIV provided partial protection against heterologous virus challenge. Pulmonary immunization with GPI-0100-adjuvanted vaccine did not induce an overt inflammatory response since influx of neutrophils and production of inflammatory cytokines were moderate and transient and lung histology was normal. Our results indicate that a GPI-0100-adjuvanted dry powder influenza vaccine is a safe and effective alternative to current parenteral vaccines.

Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response.

Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the immunoglobulin (Ig) M, IgG, and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n = 16) in comparison with healthy controls (HCs; n = 16). The Complementary Determining Region 3 region of the immunoglobulin heavy chain variable region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis, and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show interindividual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B-cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p = 0.01). Postvaccination, significant differences emerged in MGUS in relation to M-protein levels. High M-protein concentration is associated with a more oligoclonal IgG and IgA response at day 7 postvaccination, and, in contrast to HCs, vaccination also induced significant perturbations in the MGUS IgM repertoire at day 7 (p = 0.005). Monoclonal expansion in MGUS thus has an effect on the baseline B-cell repertoire and influences the recruited repertoire upon vaccination.

Specific immune responses against airway epithelial cells in a transgenic mouse-trachea transplantation model for obliterative airway disease.

BACKGROUND: Immune injury to airway epithelium is suggested to play a central role in the pathogenesis of obliterative bronchiolitis (OB) after clinical lung transplantation. In several studies, a rejection model of murine trachea transplants is used, resulting in obliterative airway disease (OAD) with similarities to human OB. To focus on the role of an immune response specifically against airway epithelium, we transplanted tracheas from transgenic mice expressing human epithelial glycoprotein (hEGP) on epithelial cells. We hypothesized that the immune response against the hEGP-2 antigen would result in OAD in the trachea transplants. METHODS: Tracheas from hEGP-2 transgenic and control nontransgenic FVB/N mice were heterotopically transplanted into FVB/N mice and harvested at week 1, 3, 6, and 9. Anti-hEGP-2 antibodies were determined in the recipient blood. The trachea grafts were analyzed for cellular infiltration, epithelial cell injury, and luminal obliteration. RESULTS: Recipients of transgenic tracheal grafts gradually developed anti-hEGP-2 antibodies. In the transgenic grafts, the submucosa was infiltrated predominantly by CD4+ T cells. Epithelial cells remained present but showed progressive abnormality. The tracheal lumen showed a mild degree of obliteration. All these changes were absent in nontransgenic FVB/N trachea transplants. CONCLUSION: The hEGP-2 antigen on the epithelial cells of transgenic trachea transplants induces specific humoral and cellular immune responses, leading to a mild form of OAD. It provides a suitable model for further investigation of the role of epithelial cells in the development of OAD in animals and OB in human-lung transplantation.

Donor brain death aggravates chronic rejection after lung transplantation in rats.

BACKGROUND: Many recipients of lung transplants from brain-dead donors develop bronchiolitis obliterans, a manifestation of chronic rejection. It has been shown that brain death increases inflammatory mediators and accelerates acute rejection in kidney, liver, and heart transplants. In this study, the authors investigated the hypothesis that brain death increases inflammatory mediators in the donor lung and subsequently aggravates chronic rejection of the lungs after transplantation in rats. METHODS: Brain death was induced in F344 rats by inflation of a subdurally placed balloon catheter. After 6 hr, donor lungs were assessed for influx of leukocytes, expression of cell adhesion molecules, and cytokine mRNA expression. For assessment of the lung after transplantation, lungs from brain-dead F344 rats were transplanted into WKY rats. Lung function after transplantation was monitored by chest radiographs during an observation period of 100 days. At the end of this period, the lungs were histologically examined; also, cytokine mRNA expression was measured. Lungs from ventilated living donors and living donors served as controls. RESULTS: After 6 hr of brain death, influx of polymorphonuclear cells and macrophages and expression of vascular cell adhesion molecule-1 in the donor lungs was increased. After transplantation at postoperative day 100, the lung function was significantly decreased compared with allografts from living donors. In the lung allografts from brain-dead donors, histologic symptoms of chronic rejection were obvious, including severe intimal hyperplasia but without bronchiolitis obliterans. Interleukin-2 mRNA was significantly increased in allografts from brain-dead donors compared with living donors. CONCLUSIONS: This study shows that brain death induces an inflammatory response in the donor lung and subsequently aggravates chronic rejection after transplantation. This may explain the clinical difference in long-term function between lungs from cadaveric donors and living donors.

Detection of alloreactive T cells by flow cytometry: a new test compared with limiting dilution assay.

BACKGROUND: Frequencies of alloreactive T cells determined by limiting dilution assays (LDA) may not adequately reflect the donor-reactive immune status in transplant recipients. To reevaluate LDA frequencies, we developed a flow cytometry test for direct determination of alloreactive T-cell frequencies and compared these frequencies with classical LDA estimates of frequencies. METHODS: For determination of frequencies by flow cytometry, peripheral blood lymphocytes (or lymphocytes taken from primary mixed lymphocyte culture) were stimulated with either Epstein-Barr virus-transformed lymphoblastoid cell lines or T cell-depleted spleen cells and stained for intracellular interferon (IFN)-gamma production and CD69. In lung transplant recipients, frequencies of IFN+ alloreactive T cells were compared with LDA frequencies, that is, cytotoxic T lymphocyte precursors and helper T lymphocyte precursors. RESULTS: With flow cytometry, alloreactive T cells were detected after overnight allostimulation as IFN-gamma CD69bright cells (range, 0.1-0.58% and 0.1-0.66% of total CD4 and CD8 cells, respectively). Frequencies increased 25-fold or more when lymphocytes were prestimulated in primary mixed lymphocyte culture before testing. After lung transplantation, mean donor-specific IFN+ CD8 T-cell frequencies did not decrease as mean donor-specific LDA cytotoxic T lymphocyte precursor frequencies, whereas no difference was seen in pretransplantation samples or third-party-specific frequencies at both time points. Mean frequencies of IFN+ CD4 did not differ from helper T lymphocyte precursors at both time points, but frequencies did not correlate. CONCLUSIONS: The flow cytometry test allows a direct measurement of alloreactive T-cell frequencies and demonstrates a discrepancy between donor-specific IFN+ CD8 T-cell frequencies and LDA CLTp after transplantation. This may be a result of the existence of "functional diverse" alloreactive T cells or of activation-induced cell death of donor-reactive T cells during long (LDA) culturing, which is avoided in the flow cytometry test.

Factors influencing functional survival of microencapsulated islet grafts.

Graft function of encapsulated islets is restricted in spite of the fact that inflammatory responses against capsules are limited to a portion less than 10%. It has been shown that dysfunction is accompanied by a gradual decrease in the glucose-induced insulin response (GIIR), a hyperproliferation of islet cells, and gradual necrosis. Also, limited survival is associated with the presence of macrophages in the overgrowth. In the present study, we investigate whether macrophages are the inducers of dysfunction of encapsulated grafts. Four weeks after successful transplantation of microencapsulated rat allografts we determined the GIIR, the rate of islet cell replication, and islet cell death. Also, we quantified the number of macrophages on the overgrown capsules. This assessment was applied to set up an in vitro coculture system of macrophages and encapsulated islets. We retrieved 93 +/- 6.2% of the capsules of which 9.2 +/- 0.3% was overgrown. The GIIR of the retrieved nonovergrown islets was reduced when compared with freshly encapsulated islets. The replication rate of the retrieved islet cells was eightfold higher than in the normal pancreas. Apoptosis was rarely observed but 37 +/- 4% of the total islet surface was composed of necrosis. We found a mean of 1542 +/- 217 macrophages per capsule. Coculture of 1500 NR8383 macrophages per encapsulated islets induced a substantial reduction in GIIR but a decrease instead of increase in replication. Necrosis was restricted to 13 +/- 1.3% of the islet cells and was not increased by the presence of macrophages. Our observations indicate that we should focus on reduction of macrophage activation and on improving the nutrition of encapsulated islets to prevent islet cell death.

Individual human serum differs in the amount of antibodies with affinity for pig fetal ventral mesencephalic cells and the ability to lyse these cells by complement activation.

Xenografting pig fetal ventral mesencephalic (pfVM) cells to repair the dopamine deficit in patients with Parkinson's disease is the focus of both experimental and clinical investigations. Although there have been marked advances in the experimental and even clinical application of these xenogeneic transplantations, questions regarding the host's xenospecific immune response remain unanswered. It has been shown that human serum is able to lyse pfVM tissue by both anti-gal-gal and non-anti-gal-gal antibodies by complement activation. The aim of this study was to investigate whether interindividual differences exist in the levels of pfVM cell-specific IgM and IgG subclass antibodies, their ability to lyse pfVM cells in vitro and the relationship between both. Pig fetal VM cells were incubated with heat-inactivated serum from 10 different individuals and binding of IgM antibodies and IgG subclass antibodies to pfVM cells was analyzed by flow cytometry. The ability to lyse pfVM cells was analyzed exposing 51Cr-labeled pfVM cells to fresh serum or isolated IgM and IgG from the same individuals and subsequent determination of released 51Cr from lysed cells. Strong differences were found between individuals in the levels of pfVM cell-specific IgM antibodies: antibody levels differed up to 40-fold. pfVM-specific IgG1 and IgG2 levels were only detectable in a few individuals. The ability to lyse pfVM cells ranged from negligible lysis up to 66.5% specific lysis. There was a strong correlation between the levels of individual pfVM-specific IgM antibodies and the ability to lyse pfVM cells in vitro. Isolated IgM, but not IgG, was able to lyse pfVM cells in the presence of complement. In conclusion, the interindividual differences in the levels of IgM with affinity for pfVM cells and their ability to lyse pfVM cells in vitro are considerable. Only few individuals possessed IgG1 and IgG2 subclass antibodies with affinity for pfVM. These findings may influence patient selection for porcine transplants and chances of graft survival in individual patients.

The role of alloresponsive Ly49+ NK cells in rat islet allograft failure in the presence and absence of cytomegalovirus.

There are still many factors to discover to explain the low success rates of islet allografts. In this study, we demonstrate that specific subpopulations of alloreactive NK cells may be involved in the failure of islet allografts. By performing allotransplantation in rats (n = 13), we observed peripheral expansion and infiltration of alloreactive Ly49i2(+) NK cells in the grafts. An effective strategy in rats to enhance the expansion of Ly49i2(+) NK cells is performing a rat cytomegalovirus infection (n = 6). Cytomegalovirus infection was associated with an early expansion of the Ly49i2(+) NK cells and accelerated islet graft failure. The Ly49i2(+) NK cells are both alloreactive and involved in virus clearance. The expansion of this subpopulation could not be blocked by cyclosporin A immunosuppression. Also alloreactive KLRH1(+) NK cells infiltrated the grafts, but nonalloreactive NKR-P1B(+) cells were not observed in the islet allografts. Perforin staining of the infiltrating NK cells demonstrated the cytotoxic capacity of these cells. Our data suggest a role for this NK subpopulation in rat islet allograft destruction.

Evaluation of monophosphoryl lipid A as adjuvant for pulmonary delivered influenza vaccine.

Prophylaxis against influenza could be improved by the development of a stable, easy to deliver, potent mucosal vaccine. In this study, we spray-freeze-dried (SFD) whole inactivated virus influenza vaccine (WIV) alone or supplemented with monophosphoryl lipid A (MPLA) using inulin as a lyoprotectant. Physical characterization revealed that the SFD powder consisted of highly porous particles with a size distribution suitable for pulmonary administration. The receptor-binding properties of WIV and the immunostimulatory properties of MPLA were preserved after spray-freeze-drying as indicated by unchanged hemagglutination titers and a retained ability of the vaccine to activate NFkB after incubation with a reporter cell line, respectively. Pulmonary vaccination of mice with MPLA-adjuvanted liquid or powder WIV resulted in induction of higher mucosal and systemic antibody concentrations than vaccination with non-adjuvanted formulations. When exposed to influenza virus, mice immunized with MPLA-adjuvanted pulmonary vaccine showed similar protection in terms of reduction in lung virus titers and prevention of weight loss as mice immunized intramuscularly with subunit vaccine. Characterization of the antibody response revealed a balanced IgG2a-to-IgG1 profile along with induction of both memory IgA- and IgG-producing B cells in mice immunized with MPLA-adjuvanted vaccine. These studies suggest that the mucosal and systemic immune responses to pulmonary delivered influenza vaccines can be significantly enhanced by using MPLA as adjuvant. MPLA-adjuvanted SFD vaccine was particularly effective implying that delivery of adjuvanted vaccine powder to the lungs can be an attractive way of immunization against influenza.

Altered cellular and humoral immunity to varicella-zoster virus in patients with autoimmune diseases.

OBJECTIVE: Patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (Wegener's) (GPA) have a 3-20-fold increased risk of herpes zoster compared to the general population. The aim of this study was to evaluate if susceptibility is due to decreased levels of cellular and/or humoral immunity to the varicella-zoster virus (VZV). METHODS: A cross-sectional study of VZV-specific immunity was performed in 38 SLE patients, 33 GPA patients, and 51 healthy controls. Levels of IgG and IgM antibodies to VZV were measured using an in-house glycoprotein enzyme-linked immunosorbent assay (ELISA). Cellular responses to VZV were determined by interferon-γ (IFNγ) enzyme-linked immunospot (ELISpot) assay and carboxyfluorescein succinimidyl ester (CFSE) dye dilution proliferation assay. RESULTS: Levels of IgG antibodies to VZV were increased in SLE patients as compared to healthy controls, but levels of IgM antibodies to VZV were not. Antibody levels in GPA patients did not differ significantly from levels in healthy controls. In response to stimulation with VZV, decreased numbers of IFNγ spot-forming cells were found among SLE patients (although not GPA patients) as compared to healthy controls. Proliferation of CD4+ T cells in response to stimulation with VZV was decreased in SLE patients but not GPA patients. CONCLUSION: SLE patients have increased levels of IgG antibodies against VZV, while cellular immunity is decreased. In GPA patients, antibody levels as well as cellular responses to VZV were comparable to those in healthy controls. These data suggest that increased prevalence of herpes zoster in SLE patients is due to a poor cellular response. Vaccination strategies should aim to boost cellular immunity against VZV.

Evaluation of an intranasal virosomal vaccine against respiratory syncytial virus in mice: effect of TLR2 and NOD2 ligands on induction of systemic and mucosal immune responses.

INTRODUCTION: RSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2) ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine. OBJECTIVE: To explore if intranasal (IN) immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity. METHODS: We produced RSV-virosomes carrying TLR2 (Pam3CSK4) and/or NOD2 (L18-MDP) ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice. RESULTS: Incorporation of Pam3CSK4 and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting) cells in vitro, as demonstrated by NF-κB induction. In vivo, incorporation of Pam3CSK4 in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam3CSK4-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam3CSK4/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced disease. CONCLUSION: Mucosal immunization with RSV-virosomes, supplemented with incorporated TLR2- and/or NOD2-ligands, represents a promising approach to induce effective and safe RSV-specific immunity.