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The membrane displayed antigen haemagglutinin (HA) from several influenza strains were expressed in the Leishmania tarentolae system. This non-conventional expression system based on a parasite of lizards, can be readily propagated to high cell density (>10(8)cells/mL) in a simple incubator at 26°C. The genes encoding HA proteins were cloned from six influenza strains, among these being a 2009 A/H1N1 pandemic strain from swine origin, namely A/California/07/09(H1N1). Soluble HA proteins were secreted into the cell culture medium and were easily and successfully purified via a His-Tag domain fused to the proteins. The overall process could be conducted in less than 3 months and resulted in a yield of approximately 1.5-5mg of HA per liter of biofermenter culture after purification. The recombinant HA proteins expressed by L. tarentolae were characterized by dynamic light scattering and were observed to be mostly monomeric. The L. tarentolae recombinant HA proteins were immunogenic in mice at a dose of 10μg when administered twice with an oil-in-water emulsion-based adjuvant. These results suggest that the L. tarentolae expression system may be an alternative to the current egg-based vaccine production., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The influence of age on the generation and persistence of specific memory B cells after vaccination with Neisseria meningitidis type C polysaccharide (MenC-PS) conjugate is unknown. MenC-PS-specific B cells could be directly enumerated by fluorochrome-labeled MenC-PS and flow cytometry in blood up to at least 4 years after vaccination, ranging from 0.01% to 0.78% of total B cells and did not correlate with age at vaccination. The percentage of MenC-specific memory B cells out of total memory B cells correlated with total MenC-specific B-cells and with frequencies of IgA(+) plus IgG(+) MenC-specific AbSC, but not with MenC-specific Ab., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Immunization with a tetanus-protein (TT) pneumococcal polysaccharide (PPS) conjugate vaccine (Pnc1-TT) induces protective immunity against lethal pneumococcal infections in neonatal and infant mice, but anti-PPS IgG response and protective efficacy is lower than in adult mice. Here, we show that reduced antibody (Ab) response and protection against infections is directly related to impaired T cell response to the carrier. Whereas spleen cells from adult mice immunized with Pnc1-TT responded with proliferation and IFN-gamma secretion to in vitro stimulation with TT, spleen cells from neonatal and infant mice did not. However, significant, but age dependent, Th2-cytokine responses were observed in mice immunized with Pnc1-TT. Impaired IFN-gamma production upon TT-stimulation in vitro was also reflected in reduced IFN-gamma/IL-5 ratio. The IL--5 response correlated with IgG anti-PPS titers, and the lack of PPS Ab in the majority of neonatal mice was clearly associated with absence of carrier-specific IL-5 production. These results show that immunization with Pnc1-TT induces carrier-specific T cell responses that increase with age and determine the levels of PPS-specific Ab elicited. Whereas a weak and Th2-biased response was observed in neonatal mice, infant mice showed a mixed Th1-Th2 response as observed in adults.

The aim of vaccination is to rapidly elicit protective immunity and generate memory for sustained protection. We studied the induction and persistence of polysaccharide (PS)-specific memory in neonatal and infant mice primed with pneumococcal conjugate (Pnc1-TT) by assessing the response to native pneumococcal PS (PPS-1), the kinetics of the PPS-1-specific IgG response to a second Pnc1-TT dose and affinity maturation. A subcutaneous (s.c.) Pnc1-TT booster induced a rapid increase in PPS-1-specific IgG, indicating efficient priming for memory by a single dose of Pnc1-TT already at 1 week of age. High levels were maintained for >12 weeks. However, a PPS-1 booster induced no response in neonatal or infant mice. The adjuvant LT-K63 significantly enhanced the IgG response and affinity to Pnc1-TT by both the s.c. and the intranasal (i.n.) route in all age groups. In neonatal and infant mice, PPS-1 and LT-K63 induced a booster response only when given i.n. following either s.c. or i.n. priming with Pnc1-TT and LT-K63. In contrast, PPS-1 with or without LT-K63 administered s.c. compromised the ongoing PPS-1-specific response elicited in neonatal mice by either s.c. or i.n. priming with Pnc1-TT and LT-K63. These results demonstrate the advantage of the mucosal route for elicitation of PS-specific memory responses in early life.

To investigate the immune response to amyloid beta-peptide (Abeta: Abeta40 and Abeta42) in peripheral human blood, sera were obtained from 36 patients with Alzheimer's disease (AD) and 34 age-matched controls. ELISA assays were used to measure antibody concentrations to Abeta-peptides. T cell response was assessed using a lymphoproliferation assay. Both AD and control subjects had low and variable concentrations of antibodies against Abeta (predominantly IgG1). The mean antibody to Abeta concentrations did not differ between groups. No specific T cell response to Abeta-peptides was detected. Natural levels of antibodies to Abeta in peripheral blood are present in all human subjects and are unlikely to be useful in the identification of patients who would respond to potential AD immune therapy. Specific cellular immune responses to Abeta in human blood were not detected.

Bordetella pertussis toxin (PTX), a key component of acellular pertussis vaccines, is known to be endowed with adjuvant properties. In experiments designed to get insights into the interactions between PTX and circulating immune cells, we first observed that addition of PTX to adult whole blood induced the release of IL-12 and TNF-alpha as well as maturation of myeloid dendritic cells (DC). These effects were still present with a toxin mutant devoid of ADP-ribosyltransferase activity but not with a formaldehyde-inactivated toxin. These findings indicate that cytokine production and DC maturation require the intact structure of PTX but not its enzymatic activity. Secondly, studies on DC generated in vitro by culturing monocytes with IL-4 and GM-CSF showed that PTX directly stimulates MHC class II and costimulatory molecules up-regulation, cytokine synthesis and NF-kappa B activation. Finally, comparison of data obtained in adult vs. cord blood revealed deficient responses of neonatal DC to PTX. These data suggest new applications of PTX and PTX mutants as vaccine adjuvants.

Pharmacologic gene regulation is a key technology, necessary to achieve safe, long-term gene transfer. The approaches described in the scientific literature all share in common the creation of artificial transcription factors by fusing a DNA-binding domain, a drug-binding domain and a transcription activation domain. These transcription factors activate the transgene expression upon binding of the pharmacologic agent (antibiotics of the tetracycline family, insect hormone, progesterone antagonist, or immunosuppressor drug) to the drug-binding domain. The major limitations to the use of these systems for human gene and cell therapies are the toxicity of the inducer molecule and the immunogenicity of the chimeric transcription factor. Thus, the gene regulation systems should operate with clinically approved drugs with safety records that do not conflict with the therapeutic gene expression regimen. This work focuses on the characterization of the immunogenicity of a tetracycline-activated transcription factor commonly used in preclinical gene therapy, rtTA2-M2, and its impact on reporter gene expression. We demonstrate that intramuscular injection of plasmid or adenoviral vectors encoding rtTA-M2 in outbred primates generates a cellular and humoral immune response to this transcription factor. The immune response to rtTA2-M2 blunts the duration of the expression the rtTA2-M2-controlled transgene in primates, presumably by destruction of the cells that coexpress rtTA2-M2 and the reporter or therapeutic gene. This immune response may result directly from the vectors used in this study, which prompts the development of new gene transfer vectors enabling safe and efficient pharmacologic gene regulation in clinic.

Although it is generally recognized that the function of the immune system declines with age, the nature of the underlying defects is still poorly understood. We now demonstrate the predominance of CD8(+)CD28(-) T cell clonal expansions in elderly persons who fail to produce specific Abs following influenza vaccination. These clones express effector cell markers and are mostly CD45RA(+). When isolated and put into culture, they are unable to proliferate, but produce IFN-gamma (but no IL-5) upon stimulation with anti-CD3 or autoantigen. These autoreactive CD8(+) type 1 effector cells seem to trigger a Th1 polarization, as CD4(+) T cells from elderly persons without in vivo Ab production produce Th1, but only low amounts of Th2 cytokines upon in vitro stimulation with PHA. Therefore, the increased occurrence of CD8(+)CD28(-) clonal expansions may be decisive for the development of immune deficiency in the elderly.

In order to study the respective roles of CD4, CD8, and CD56 (NK) cells in gamma interferon (IFN-gamma) production after in vitro stimulation with flu vaccine in a healthy adult human population, we depleted these cellular subtypes before stimulation with antigen (inactivated split vaccine, A/Texas H1N1, or A/Sydney H3N2). We observed that while CD4 cells were required for IFN-gamma secretion in both conditions in vitro, CD56 (NK) cells and, to a lesser extent, CD8 cells had a negative effect on such synthesis upon H1N1 stimulation, as judged by an increased number of spots compared to the initial undepleted population. This regulation of IFN-gamma secretion was associated with an increase in ICAM-1 expression, in particular on T and B cells. This study points out the importance of evaluating in vitro immune responses on a whole-cell population in addition to isolated subtypes if one needs to address potential cellular interactions occurring in vivo in some situations (H1N1 stimulation in the present case). Such cross-regulations occur even in vitro during the antigenic stimulation step.

We prepared a series of cationic lipid vesicles comprising a cationic cholesterol derivative, DC-Chol with or without a neutral phospholipid, DOPC or DOPE. The vesicles were tested for their ability to bind and adjuvant split inactivated influenza vaccines. We found that DC-Chol-containing liposomes are capable to strongly bind influenza vaccine antigens upon simple mixing with the vaccine. The resulting formulations induced robust anti-influenza immune responses both after s.c. and i.n. administration in BALB/c mice while neutral Cholesterol/DOPC liposomes displayed virtually no stable antigen binding and no adjuvant effect. The parenteral adjuvant effect of DC-Chol on trivalent split influenza vaccines was then confirmed in outbred mice and monkeys. Among the most potent formulations tested, a simple mixture of the vaccine with a microfluidized dispersion of DC-Chol in an aqueous buffer is being considered for further development to produce an improved influenza vaccine.

Polymorphonuclear cells (PMNs) from healthy donors and differentiated HL-60 cells were compared in an opsonophagocytic assay using fluorescent latex beads coated with Streptococcus pneumoniae polysaccharide conjugates. Serum-specific phagocytosis was efficiently mediated by both sources of cells, as measured by flow cytometry, but the mean number of beads ingested per cell was three- to fivefold higher when PMNs were used than when HL-60 cells were used. Nevertheless, differentiated HL-60 cells could be a convenient and standardized source of cells to evaluate the functionality of specific antibodies to vaccine candidates as a coating on fluorescent beads.

After primary infection in childhood, varicella zoster virus (VZV) remains latent in the dorsal route ganglia. Its reactivation later in life can lead to a zoster episode. VZV-specific, T-cell-mediated immunity (VZV-CMI) is likely to be important in preventing symptomatic reactivation. As CMI declines with age, a vaccine enhancing VZV-CMI might be effective in decreasing the incidence or severity of zoster in elderly subjects. A randomized, double blind controlled trial assessing CMI responses of elderly subjects immunized with a live attenuated, VZV-Oka vaccine was conducted. Two hundred healthy volunteers (55-75 years of age) received either a single injection of the VZV vaccine (PMC), containing 3200 (Oka 3200), 8500 (Oka 8500), or 41,650 (Oka 41650) PFU of live VZV, or a pneumococcus vaccine control group (Pneumo 23((R)). The immune response to VZV was assessed by measuring the T-cell response to VZV antigens, i.e. proliferation (stimulation index, SI), precursor cell frequency (PCF), cytokine secretion, and antibody titers. Six weeks post-vaccination, VZV-specific SI (adjusted mean values) was significantly greater (P<0.0001) in the 3 vaccine groups (with SI=5. 6 for Oka 3200; SI=5.0 for Oka 8500, and SI=7.2 for Oka 41,650) than in the control group (SI=2.9). The increase in PCF was striking, with 72.4, 91.2 and 85.1 precursors per million cells respectively in these 3 vaccine groups, vs 26.3 in the control group. No significant IL-4 secretion was observed in any subject, whereas the presence of IFN-gamma secretion was found to correlate with good responder status. The increase of these CMI parameters did not depend upon the titer of virus injected. Geometric mean titers of VZV antibodies increased in all vaccine groups and remained unchanged in the control group. Nevertheless, no correlation between the antibody response and the cell-mediated response was found. Live attenuated VZV vaccine caused a significant increase in VZV-CMI in a healthy, elderly population. No relationship between vaccine dose and the intensity of the specific response was found.

We have used spring powered jet injectors to deliver a solution of a naked DNA vaccine encoding the influenza hemagglutinin HA into the skin of mice and monkeys. We compared the immune responses induced by this needleless injection technique into the skin to the responses induced by a classical i.m. immunization. Both routes of immunization induced significant ELISA antibody titers and hemagglutination inhibition (HI) titers that were above the usual threshold values predictive of protection against influenza in mice and monkeys. In mice, both ways of immunization were equally efficient in inducing HA-specific CTL responses. Regarding antibody isotypes, the IgG1/IgG2a ratio was in favour of the IgG2a isotype for i.m. immunization and more balanced for i.d. immunization. The ability of the two injection techniques to induce immunity in mice did not correlate with transgene expression in the site of administration. In fact, local gene expression was 10-100 fold more important in the injected muscle as compared to the jet-injected skin when assessed by using the luciferase reporter system.

Decreased cell-mediated immune (CMI) response to varicella-zoster virus (VZV) is correlated with an increased risk of reactivation of latent virus from dorsal root sites, leading to herpes zoster. The cell-mediated and humoral immunogenicity of three concentrations (3200, 8500, and 41,650 pfu/dose) of a live attenuated VZV vaccine (Oka strain; VZV/Oka) was compared with a control pneumococcal polysaccharide vaccine in 200 healthy adults who were > or = 55 years old. Six weeks after vaccination, the VZV-specific CMI response (as measured by stimulation index values and precursor cell frequencies) was enhanced in all VZV/Oka vaccine groups compared with the control group (for all VZV/Oka groups combined vs. controls, tested with VZV crude antigen: stimulation index, P < .001; precursor cell frequency, P < .001). Geometric mean titers of anti-VZV antibodies increased in all VZV/Oka vaccine groups but remained unchanged in the control vaccine group. No dose effect of VZV/Oka vaccine was observed for CMI or humoral responses.

To study in mice the effects of in vivo xenogenic immunization with human major histocompatibility complex (MHC) class II antigens, the animals were injected with HLA-DR antigens and their proliferative responses tested in vitro. The results showed that small amounts of HLA-DR proteins, acting as nominal antigens, were not only able to prime mice for a secondary in vitro xenogenic mixed lymphocyte reaction but also induced a syngeneic mixed lymphocyte reaction. In contrast, allogeneic or syngeneic immunization of mice with soluble MHC class II molecules failed to stimulate an autoreactive response. The syngeneic mixed lymphocyte reaction was primarily directed against syngeneic MHC class II molecules since the murine T lymphocytes reacted against MHC class II-positive dendritic spleen cells and MHC class II-transfected mouse fibroblasts. A self-reactive T-cell line induced under these experimental conditions did not react in xenogeneic and allogeneic mixed lymphocyte reactions. However, these T lymphocytes proliferated when human peripheral blood lymphocytes of various haplotypes were presented in the context of syngeneic mouse antigen presenting cells.

Immunization of mice with the p-azobenzenearsonate-L-tyrosine conjugate (ABA-Tyr) leads to the activation of ABA-specific T helper cells capable of proliferating in vitro in the presence of the corresponding antigen. This response is under a dual genetic regulation by H-2 and non-H-2 linked genes, and H-2d mice are high-responder. We demonstrate here, using strains congenic to BALB/c for chromosomes (Chr.) 6 or 12 that this T cell response is also influenced by kappa and IgH linked genes. Double congenic mice indicate that Chr.6 and Chr.12 genes have a complementary effect on the response which cannot be predicted solely by the alleles expressed on either of the two chromosomes. In addition, responses in Bailey's inbred recombinant mice allow a possible mapping of the Chr.12 gene at the 5' end of the IgH complex and of the VH-dextran gene family. The mechanisms which may account for the influence of immunoglobulin gene products on the ABA-specific T cell repertoire are discussed.

Cytotoxic treatment of BALB/c cells from different peripheral lymphoid tissues by a cocktail of monoclonal antibodies against Thy-1, Ly-1, L3T4 and Ly-2 differentiation markers (anti-T cocktail) plus complement eliminates all mature T lymphocytes. Yet a population of dull Thy-1+, Ly-1-, L3T4-, Ly-2-, corresponding to about 1% of the initial population, can be detected by flow cytometry which proliferate under concanavalin A stimulation. These anti-T killing-resistant cells (TKR) were previously shown to be capable of differentiating in culture into class II-restricted autoreactive T helper cells. We demonstrate here that such cells can be detected in mice of BALB/c and DBA/2 genetic background but are absent in C57BL/6 and B10 animals. The presence of TKR cells is dominant in (BALB/c x C57BL/6)F1 hybrids and genetically controlled by two genes which are neither H-2 nor Igh linked. TKR cells are also detected in young NZB mice but disappear with the development of the systemic autoimmune disease in old animals. Thy-1+, L3T4-, Ly-2- cells from MRL lpr/lpr mice also respond to concanavalin A but are removed by the anti-T treatment. Altogether, arguments are presented suggesting that TKR cells represent a particular subset of double-negative peripheral T cells which may correspond to autoreactive T cell recursors that would escape the thymic selection. We postulate that these cells are present in all mouse strains but their susceptibility to killing by anti-Thy-1 antibodies differs depending on background genes.

The T cell repertoire of BALB/c mice contains clones capable of recognizing p-azobenzenearsonate (ABA)-tyrosine (Tyr) in association with both I-A and I-E-encoded class II molecules. Immunization of BALB/c animals with ABA-GAT (terpolymer of L-Glu60-L-Ala30-L-Tyr10) or ABA-GLT (terpolymer of L-Glu51-L-Lys34-L-Tyr15) instead of ABA-Tyr reduces the secondary proliferative response to ABA-Tyr in vitro. Limiting dilution experiments indicate that this situation corresponds to the recruitment of fewer ABA-specific T cells in vivo. The same experiments, performed in A.TH mice, which are nonresponder to both GAT and GLT, demonstrate that the number of ABA-specific T cells stimulated in vivo with ABA conjugates depends on the Ir gene-controlled immunogenicity of the carrier rather than on the ABA epitope density on the immunogen. Although GAT is preferentially recognized in association with A, and GLT with E, ABA-GAT and ABA-GLT stimulate both A and E-restricted ABA T cells in vivo and in vitro. The ABA-Tyr-specific T cell repertoire is not qualitatively affected by the carrier. This demonstrates that the inhibition of hapten-specific T cell expression upon immunization with ABA conjugates does not result from a competition between hapten and carrier-specific T cells for epitope recognition in association with the same Ia molecule on antigen-presenting cells.

The T lymphocyte repertoire consists of clones recognizing foreign antigens together with self histocompatibility molecules. Diversification of the receptor is believed to arise by somatic mechanisms during ontogeny. MHC gene products are essential for this process as well as for antigen recognition and expression of T cell functions. Yet, the antigen-specific T cell receptor is not encoded by MHC genes. Little is still known concerning the nature and the genetic origin of this receptor despite numerous experimental approaches. Although the T cell repertoire is mainly determined, in a single individual, by the alleles expressed at the MHC locus, one can postulate that it could also be influenced by the existence of alleles of the germ line gene(s) encoding the T cell receptor. If so, an analysis of the T cell fine specificity in mice of the same H-2 haplotype with different background genes might permit the mapping of the genes coding for this receptor. Such an experimental approach requires the use of an antigen consisting of only one major determinant. Several recent observations suggested to us that the hapten p-azobenzenearsonate (ABA) was a suitable model for such investigations. Thus, we decided to compare the specific pattern of responses to ABA-tyrosine, ABA-histidine and to free ABA in different inbred mouse strains. We report here that the lymph node T cell proliferative response to these molecules is under the control of an ABA-specific Ir gene. The ABA-Tyr conjugate is the most potent immunogen of the three in vivo as well as in vitro. High responder strains to ABA-His or ABA are included in the group of high responders to ABA-Tyr suggesting that the response to the three molecules is under the control of the same Ir-gene. The pattern of the response is also influenced by background gene(s). One of these can be localized on chromosome 12 using congenic mice. No close linkage to IgCH markers or VH idiotypes can be demonstrated but a linkage of this gene(s) to the Pre-1 locus seems possible. B lymphocytes do not seem to account for the involvement of Chr.12-genes in the response since; in our experimental system, they do not present ABA to T cells nor do they proliferate in the assays. Similarly, ABA-Tyr-antibody complexes do not enhance macrophages presentation of ABA to T cells, which supports the conclusion that IgCH or VH gene products are not involved in the control of the response.(ABSTRACT TRUNCATED AT 400 WORDS)