Role of complement receptor type 2 and endogenous complement in the humoral immune response to conjugates of complement C3d and pneumococcal serotype 14 capsular polysaccharide

Conjugation of the complement fragment C3d to both T-cell-dependent (TD) protein and T-cell-independent type 2 (TI-2) polysaccharide antigens enhances the humoral immune response in mice immunized with either type of antigen. However, the ability of C3d-protein conjugates to enhance the antibody response in mice deficient in complement receptor types 1 and 2 (CR1 and CR2) has raised questions about the role of C3d-CR2 interactions in the adjuvant effect of C3d. In this study, we examined the role of CR2 binding and endogenous complement activation in the antibody response to conjugates of C3d and serotype 14 pneumococcal capsular polysaccharide (PPS14). To block binding of PPS14-C3d conjugates to CR2, mice were immunized with a mixture of vaccine and (CR2)2-immunoglobulin G1 (IgG1). Mice receiving (CR2)2-IgG1 at the time of primary immunization had a marked reduction in the primary anti-PPS14 antibody response but an enhanced secondary anti-PPS14 response, suggesting that C3d-CR2 interactions are required for the primary response but can have negative effects on the memory response. Further, compared with mice receiving PPS14-C3d having a high C3d/PPS14 ratio, mice immunized with PPS14-C3d with low C3d/PPS14 ratios had an enhanced secondary antibody response. Treatment of mice with cobra venom factor to deplete complement had insignificant effects on the antibody response to PPS14-C3d. Experiments with CBA/N xid mice confirmed that PPS14-C3d conjugates retain the characteristics of TI-2 rather than TD antigens. Thus, the adjuvant effect of C3d conjugated to PPS14 requires C3d-CR2 interactions, does not require activation of endogenous complement, and is not mediated by TD carrier effects.     

Increased immunogenicity and induction of class switching by conjugation of complement C3d to pneumococcal serotype 14 capsular polysaccharide

Previous studies have demonstrated an adjuvant effect for the C3d fragment of complement C3 when coupled to T-dependent protein antigens. In this study, we examined the antibody response to covalent conjugates of C3d and a T-independent antigen, the capsular polysaccharide of serotype 14 Streptococcus pneumoniae (PPS14). We prepared a conjugate of mouse C3d and PPS14 and compared its immunogenicity with that of a conjugate of PPS14 and ovalbumin (OVA). When BALB/c mice were immunized with PPS14-C3d, there was a significant increase in serum anti-PPS14 concentrations compared with either native PPS14 or control PPS14-glycine conjugates. This was accompanied by a switch in anti-PPS14 from predominantly immunoglobulin M (IgM) to IgG1 by day 25 following primary immunization. Following secondary immunization with PPS14-C3d, there was a marked booster response and a further increase in the ratio of IgG1 to IgM anti-PPS14. Although the primary antibody response to the PPS14-OVA conjugate exceeded that induced by immunization with PPS14-C3d, serum anti-PPS14 concentrations after a second injection of PPS14-C3d were nearly identical to those induced by secondary immunization with PPS14-OVA. Experiments with athymic nude mice suggested that T cells were not required for the adjuvant effect of C3d on the primary immune response to PPS14 but were necessary for enhancement of the memory response after a second injection of PPS14-C3d. These studies show that the adjuvant effects of C3d extend to T-independent antigens as well as T-dependent antigens. As a means of harnessing the adjuvant potential of the innate immune system, C3d conjugates may prove useful as a component of vaccines against encapsulated bacteria.     

Immunization of aged mice with a pneumococcal conjugate vaccine combined with an unmethylated CpG-containing oligodeoxynucleotide restores defective immunoglobulin G antipolysaccharide responses and specific CD4+-T-cell priming to young adult levels

Polysaccharide (PS)-protein conjugate vaccines, in contrast to purified PS vaccines, recruit CD4+-T-cell help and restore defective PS-specific humoral immunity in the immature host. Surprisingly, in the immunocompromised, aged host, anti-PS responses to conjugate vaccines are typically no better than those elicited by purified PS vaccines. Although aging leads to defects in multiple immune cell types, diminished CD4+-T-cell helper function has recently been shown to play a dominant role. We show that in response to immunization with purified pneumococcal capsular PS serotype 14 (PPS14) in saline, the T-cell-independent immunoglobulin G (IgG) anti-PPS14 response in aged mice was comparable to that in young mice. In contrast, the T-cell-dependent IgG anti-PPS14 response to a soluble conjugate of PPS14 and pneumococcal surface protein A (PspA) (PPS14-PspA) in saline was markedly defective. This was associated with defective priming of PspA-specific CD4+ T cells. In contrast, immunization of aged mice with PPS14-PspA combined with an unmethylated CpG-containing oligodeoxynucleotide (CpG-ODN) restored IgG anti-PPS14 responses to young adult levels, which were substantially higher than those observed using purified PPS14. This was associated with enhanced PspA-specific CD4+-T-cell priming. Similarly, intact Streptococcus pneumoniae capsular type 14, which contains Toll-like receptor (TLR) ligands, also induced substantial, though modestly reduced, T-cell-dependent (TD) IgG ant-PPS14 responses in aged mice. Spleen and peritoneal cells from aged and young adult mice made comparable levels of proinflammatory cytokines in response to CpG-ODN, although cells from aged mice secreted higher levels of interleukin-10. Collectively, these data suggest that inclusion of a TLR ligand, as an adjuvant, with a conjugate vaccine can correct defective TD IgG anti-PS responses in elderly patients by augmenting CD4+-T-cell help.     

Antibody Response to Pneumococcal Polysaccharide Vaccine in Young, Adult and Old Mice

The anti-pneumococcal antibody response was studied in young (5-week-old) and adult (10-week-old) BALB/c and CBA/J mice and in adult (9–10-week-old) and old (12-, 18- and 24-month-old) AB6F₁ and B6D2F₁ mice after s.c. immunization with a 23-valent pneumococcal polysaccharide vaccine. Both young and adult mice showed a significant IgM antibody response to the vaccine 6 days after immunization with 111 /ig antigen. There were significant immune responses to serotypes 1, 2, 4 and 7F in contrast to small responses to serotypes 14, 19F and 23F after immunization with the vaccine. One month after immunization, there were only marginal differences in IgM anti-pneumococcal antibody levels to the vaccine (anti-PPS) between immunized and unimmunized BALB/c mice, whereas in CBA/J mice the anti-PPS remained higher in immunized than in unimmunized mice. Immunization of old mice induced a significant IgM antibody response 6 days after immunization, but the anti-PPS thereafter decreased rapidly towards preimmunization values in AB6F₁ mice. A significant IgG anti-PPS was not detected in any of the mice studied. The IgA anti-PPS tended to vary over time with no consistent pattern. It is important to carefully consider age and strain of the mice used when studying the immune response to pneumococcal polysaccharide antigens.     

肺炎结合疫苗PPS14-OVA、PPS14-C3d在小鼠脾脏的定位及补体对定位的影响

目的检测血清型14型肺炎球菌荚膜多糖(PPS14)结合疫苗PPS14-OVA(卵清蛋白)、PPS14-C3d(补体)免疫小鼠后在脾脏细胞中的定位,以及补体对其定位的影响。方法PPS14-OVA、PPS14-C3d经皮下或静脉注射,免疫正常小鼠和眼镜蛇毒因子(CVF)预处理的小鼠,应用免疫荧光技术检测脾脏冰冻切片中的PPS14及各种免疫细胞。结果正常小鼠免疫PPS14-OVA后,PPS14-OVA定位于脾脏;1 h后与脾脏边缘区B细胞有结合;2 h后出现在淋巴滤泡中;6 h后全部转移至淋巴滤泡,并与滤泡B细胞和树突状细胞结合,与巨噬细胞和T细胞没有结合。而CVF预处理的小鼠,PPS14-OVA不定位于脾脏。PPS14-C3d在脾脏中的定位不受CVF处理的影响。结论 PPS14-OVA在脾脏中的定位需要补体的介导,而PPS14-C3d不需要。     

The role of complement in the induction of antibody responses.

To determine the effect of complement on the normal antibody response to T cell-dependent antigens, we immunized normal and C4 deficient guinea-pigs with bacteriophage phi X 174. Following primary immunization with a standard dose (2 X 10(9) PFU/Kg) given intravenously. C4 deficient guinea-pigs produced less antibody than normal guinea-pigs and were unable to maintain measurable antibody levels. Following secondary immunization, antigen clearance of C4 deficient guinea-pigs was delayed and the subsequent antibody response was identical to their primary response without amplification or isotype switch. Increased antigen dose and administration of antigen in adjuvants into footpads improved the responses but did not make them normal. The primary and secondary responses became essentially normal, however, when small amounts of normal guinea-pig serum were given to the deficient animals at the time of the primary (but not the secondary) immunization. We postulate that the contribution of complement to the mature humoral immune response is related to activation of C3. Our data show that antigen initiates a primary immune response. The resultant antigen-antibody complexes interact with complement and are then non-specifically trapped by C3 receptors on dendritic cells, B cells and macrophages. Thus, antigen is selectively accumulated within the lymphoid organs and in turn 'captures' antigen specific B cells by interaction of the trapped antigen with antigen specific sIg. The approximation of specific lymphoid cells, macrophages and antigen permits generation of specific memory cells and ensures prompt, mature antibody response on subsequent antigen exposure.     

Effect of Mucosal and Systemic Immunization with Pneumococcal Polysaccharide Type 3, 4 and 14 in the Rat

Four immunization routes were investigated to induce an immune response against three structurally different types of pneumoccoccal polysaccharide (PPS) in the rat. In particular, the contribution of the IgA isotype in these immune responses was studied. Six days after administration of PPS type 3, 4 or 14, the localization of specific antibody-containing cells (ACC) in different lymphoid tissues and the antibody titres in serum were studied. All four routes induced anti-PPS ACC in the spleen. After intraduodenal, intravenous and especially intraperitoneal administration of PPS, many IgA-specific anti-PPS ACC were also found in parathymic and mesenteric lymph nodes and in the lamina propria of intestinal tissue. Several anti-PPS ACC were found in Peyer's patches, located peripheral of the B-cell areas. The intratracheal immunization elicited only a local immune response, in bronchus-associated lymphoid tissues and paratracheal lymph nodes. The localization of these anti-PPS ACC was influenced by the route of immunization. After all four investigated routes, specific antibodies were found in serum against PPS. However, some remarkable differences between PPS-3, 4 and 14 were found in the magnitude of the immune response and the distribution of the isotypes. Both route of immunization and structure of the PPS have a profound influence on the immune responses in rats.     

Complement, membrane glycoproteins, and complement receptors: their role in regulation of the immune response

To determine the effect of complement on the normal antibody response we studied seven patients with genetically determined complement component deficiencies, guinea pigs deficient of C4 and C2, respectively, and two patients whose neutrophils and monocytes lack the C3bi receptor. Patients deficient of early complement components (C4, C2, C3) have abnormal antibody responses to the T-cell-dependent antigen, bacteriophage phi X 174. Complement-deficient guinea pigs (C4, C2) produce less antibody than normal guinea pigs and are unable to maintain measurable antibody levels; during secondary immunization they fail to develop amplification and to switch from IgM to IgG. This defect can be overcome by increasing the antigen dose or by injecting normal guinea pig serum at the time of the primary (but not the secondary) immunization. Patients with deficiency of the C3bi receptor were shown to have a significantly depressed antibody response to T-dependent antigens. We postulate that the contribution of complement to the mature humoral immune response is related to activation of C3. The initial production of IgM following antigen injection leads to antigen-antibody complexes which interact with complement, to be nonspecifically trapped by C3b and C3bi receptors on B cells or macrophages. Thus antigen is selectively accumulated within the lymphoid organs and in turn may entrap antigen-specific B cells by interaction of the trapped antigen with surface immunoglobulin. As a result, close approximation between antigen, antibody, and a network of specific and nonspecific lymphoid cells is initiated, allowing generation of specific memory cells and initiation of a prompt mature antibody response on subsequent exposure to antigen.     

Hemolysin-producing Listeria monocytogenes affects the immune response to T-cell-dependent and T-cell-independent antigens.

A murine experimental infection with a hemolysin-producing (Hly+) strain of Listeria monocytogenes and a non-hemolysin-producing (Hly-) mutant was used as an in vivo model to evaluate the role of hemolysin production in the immune response. No antilisterial antibodies were detectable following sublethal infection with Hly+ bacteria, but consistent antilisterial immunoglobulin G (IgG) and IgM antibody production was observed following sublethal infection with the Hly- mutant. Hly+ but not Hly- L. monocytogenes induced transient inhibition of antibody response to Hly- bacteria and to unrelated T-cell-dependent (tetanus toxoid) and T-cell-independent (pneumococcal polysaccharide 3) antigens. Transient inhibition of the activation of an antigen-specific T-cell clone was also observed following Hly+ infection of antigen-presenting cells but not following Hly- infection. These results suggest that hemolysin production by L. monocytogenes is an important factor in modulating the immune response to T-cell-dependent and T-cell-independent antigens in infected individuals.     

Tolerance to self gangliosides is the major factor restricting the antibody response to lipopolysaccharide core oligosaccharides in Campylobacter jejuni strains associated with Guillain-Barré syndrome

Guillain-Barré syndrome following Campylobacter jejuni infection is frequently associated with anti-ganglioside autoantibodies mediated by molecular mimicry with ganglioside-like oligosaccharides on bacterial lipopolysaccharide (LPS). The regulation of antibody responses to these T-cell-independent antigens is poorly understood, and only a minority of Campylobacter-infected individuals develop anti-ganglioside antibodies. This study investigates the response to gangliosides and LPS in strains of mice by using a range of immunization strategies. In normal mice following intraperitoneal immunization, antibody responses to gangliosides and LPS are low level but can be enhanced by the antigen format or coadministration of protein to recruit T-cell help. Class switching from the predominant immunoglobulin M (IgM) response to IgG3 occurs at low levels, suggesting B1-cell involvement. Systemic immunization results in poor responses. In GalNAc transferase knockout mice that lack all complex gangliosides and instead express high levels of GM3 and GD3, generation of anti-ganglioside antibodies upon immunization with either complex gangliosides or ganglioside-mimicking LPS is greatly enhanced and exhibits class switching to T-cell-dependent IgG isotypes and immunological memory, indicating that tolerance to self gangliosides is a major regulatory factor. Responses to GD3 are suppressed in knockout mice compared with wild-type mice, in which responses to GD3 are induced specifically by GD3 and as a result of polyclonal B-cell activation by LPS. The anti-ganglioside response generated in response to LPS is also dependent on the epitope density of the ganglioside mimicked and can be further manipulated by providing secondary signals via lipid A and CD40 ligation.     

Early life T cell responses to pneumococcal conjugates increase with age and determine the polysaccharide-specific antibody response and protective efficacy

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.     

Effect of Boar Seminal Immunosuppressive Component on Humoral Immune Response in Mice

PROBLEM: The effect of seminal immunosuppressive component (ISF) on the primary and secondary antibody response, induced by soluble and/or corpuscular antigens, was evaluated in the sera obtained at different intervals before and after immunizations. The duration of the immune suppression induced by ISF treatment within the primary and secondary immunizations was also determined. METHOD OF STUDY: The ability of the seminal immunosuppressive component to suppress the primary and secondary antibody response was evaluated by enzyme-linked immunoadsorbent assay (ELISA) in the sera of mice treated in vivo with the immunosuppressor before and after immunization with antigens. Likewise, the duration of the immune suppression induced by the seminal immunosuppressor administered before the primary and secondary immunizations was tested by ELISA with antisera to keyhole limpets hemocyanin. RESULTS: Intravenous and rectal deposition of ISF led to a suppression of the primary and secondary antibody response to soluble and corpuscular antigens. The most effective suppression of the immune response was achieved in mice treated with immunosuppressor 3 days before the immunization with antigens. Also the secondary antibody response to the challenging antigen was significantly suppressed by ISF. The production of immunoglobulin G (IgG), IgM, and IgA to keyhole limpets hemocyanin was depressed for a relatively long period in mice treated with the immunosuppressor. The results indicated that the preexposure is needed for maximal immunosuppression of the primary antibody production. The treatment with ISF led to a prolonged immunosuppression but not to permanent tolerance to the challenging antigen. CONCLUSIONS: The in vivo deposition of semen may compromise some aspects of the immune system and may be an important factor in the development of viral and bacterial infections. The suppression of humoral immune response suggests potential uses of seminal immunosuppressor for the animal model study in the therapy of antibody-mediated diseases.     

Antigenic competition in IgE antibody production. I. Establishment of parameters involved in primary and secondary responses.

Antigenic competition was demonstrated in IgE antibody response in mice immunized with ovalbumin or DNP-ovalbumin associated with several non-related proteins: DNP-Ascaris, DNP-keyhole limpet haemocyanin or Ascaris. Simultaneous injection of two antigens caused a suppression of IgE antibody production to the test antigen, IgG1 antibody formation being only diminished under certain conditions. Competition was dose-dependent and effective only in the primary response. However, the secondary response could be also partially suppressed if the competitor antigen was given in both first and second antigenic stimulation. Competition was abolished by irradiation prior to immunization.     

Immunization of female mice with glycoconjugates protects their offspring against encapsulated bacteria

The immune system of the newborn is immature, and therefore it is difficult to induce protective immunity by vaccination in the neonatal period. Immunization of mothers during pregnancy against infections caused by encapsulated bacteria could thus be particularly attractive, as infants do not respond to polysaccharide (PS) antigens. Transmission of maternal vaccine-specific antibodies and protection of offspring against pneumococcal bacteremia and/or lung infection were studied in a neonatal murine model of pneumococcal immunization and infections. Adult female mice were immunized with native pneumococcal PS (PPS) of serotypes 1, 6B, and 19F or PPS conjugated to tetanus protein (Pnc-TT), and PPS-specific antibodies were measured in sera of mothers and their offspring. Effective transmission of maternal antibodies was observed, as PPS-specific immunoglobulin G levels in 3-week-old offspring of immunized mothers were 37 to 322% of maternal titers, and a significant correlation between maternal and offspring antibody levels was observed. The PPS-specific antibodies persisted for several weeks but slowly decreased over time. Offspring of Pnc-TT-immunized mothers were protected against pneumococcal infections with homologous serotypes, whereas PPS immunization of mothers did not protect their offspring, in agreement with the low titer of maternal PPS specific antibodies. When adult female mice were immunized with a meningococcal serogroup C conjugate vaccine (MenC-CRM), antibody response and transmission were similar to those observed for pneumococcal antibodies. Importantly, bactericidal activity was demonstrated in offspring of MenC-CRM-immunized mothers. These results demonstrate that this murine model of pneumococcal immunization and infections is suitable to study maternal immunization strategies for protection of offspring against encapsulated bacteria.     

Increased adjuvant efficacy in stimulation of antibody responses after macrophage elimination in vivo.

To investigate whether macrophages influence the efficacy of adjuvants, we locally eliminated lymph node macrophages with dichloromethylene diphosphonate containing-liposomes before primary immunization. After macrophage elimination, animals were immunized with a soluble antigen (TNP-KLH; 2,4,6-trinitrophenyl-keyhole limpet haemocyanin) either in phosphate-buffered saline (PBS), in complete Freund's adjuvant (CFA), or in Specol is a water-in-oil emulsion, considered to be less aggressive than CFA, but equally effective. A secondary immunization followed with TNP-KLH. In vivo macrophage elimination before Specol/TNP-KLH immunization resulted in increased adjuvant efficacy as measured by (primary) antibody responses. This suggests that the activity of Specol is not primarily mediated through macrophages but rather through other antigen-presenting cell types (e.g. dendritic cells, B cells, fibroblasts). The overall quality of produced antibodies, in terms of isotype distribution and affinity maturation, remained the same. After primary injection, CFA/TNP-KLH immunization resulted in significantly higher antibody responses in macrophage-depleted animals and antibody responses did not increase significantly after secondary immunization. However, a booster effect was found when macrophages were not eliminated before CFA/TNP-KLH immunization, suggesting that the presence of macrophages during the first weeks of the primary immune response is essential for the induction of an effective secondary response in CFA immunizations. In conclusion, macrophage depletion before immunization with a soluble T-cell-dependent antigen combined with Specol may enhance specific antibody responses without changing the isotype or affinity of the antibodies.     

Inhibition of the neutrophilic infiltrate of experimental tubulointerstitial nephritis in the Brown-Norway rat by decomplementation

Anti-tubular basement membrane (TBM) antibody-associated tubulointerstitial nephritis (TIN) in Brown-Norway rats is induced by immunization with bovine TBM antigens and adjuvants. The lesion is characterized by linear deposition of IgG and C3 along the TBM with sequential neutrophil (Days 8-9)- and mononuclear (Day 10 and after)-dominated inflammatory infiltrates. To study the complement dependence of the infiltrative process, immunized rats were decomplemented with cobra venom factor (CVF). The CVF treatment did not affect the production or renal deposition of anti-TBM antibodies. CVF markedly reduced the neutrophilic inflammatory infiltrate. In rats immunized with suboptimal doses of soluble bovine TBM antigens to produce a mild lesion, decomplementation also decreased the mononuclear inflammatory infiltrates on Days 10-13. In rats immunized optimally with particulate TBM to induce maximally severe TIN, decomplementation did not affect the mild mononuclear cell infiltrate on Days 8 and 9 but did somewhat reduce the subsequent mononuclear infiltrate on Days 10 and 12. These results demonstrate that the anti-TBM antibody- and C3-associated neutrophilic inflammatory infiltrate is largely complement dependent. The early mononuclear cell infiltrate that was unmodified by CVF treatment may be dependent on complement-independent humoral events or related to cell-mediated immune events. A portion of the later mononuclear inflammatory infiltrate could be dependent on the preceding neutrophilic inflammatory phase.     

Alveolar macrophages down-regulate local pulmonary immune responses against intratracheally administered T-cell-dependent, but not T-cell-independent antigens.

The role of alveolar macrophages in the pulmonary immune response against various antigens was studied after elimination of alveolar macrophages by intratracheal administration of liposome-encapsulated dichloromethylene diphosphanate. When the responses against T-cell-independent type 1 and type 2 antigens were compared, it was found that elimination of alveolar macrophages had no effect on T-cell-independent antigens. Intratracheal antigen administration resulted in low lung associated, local responses, although some response was observed in the spleen. In contrast, elimination of alveolar macrophages resulted in an increase in local pulmonary immune response against T-cell-dependent antigens. We conclude from these experiments that alveolar macrophages play an important role in controlling the local pulmonary immune response against T-cell-dependent antigens by down-regulation of local T-cell populations. The alveolar macrophages do not down-regulate the response against intratracheally administered T-cell-independent antigens, although they are important in the protection against inflammatory damage caused by bacterial endotoxins.     

The glucuronoxylomannan of Cryptococcus neoformans serotype A is a type 2 T-cell-independent antigen.

The humoral immune response of inbred mice to immunization with the glucuronoxylomannan (GXM) of Cryptococcus neoformans was investigated both serologically and in plaque-forming cells (PFCs). The T-helper-cell-independent quality of the GXM was demonstrated by using BALB/c nu/nu mice. Primary and secondary dose responses to three antigenic forms of GXM, (i) the native antigen, (ii) a GXM-bovine serum albumin protein conjugate, and (iii) a cryptococcal whole-cell vaccine, revealed a lack of isotype class switching and anamnestic responses. Both the levels of complement-fixing anti-GXM antibody in serum and the PFC responses in the athymic mice showed no significant differences from those in the wild-type controls. However, T cells are involved in the suppression of the primary response to GXM. When BALB/cBy mice were given rabbit anti-mouse thymocyte serum along with 0.5 microgram of GXM, both antibody levels in serum and PFC responses were significantly increased over those of control mice that received GXM and normal rabbit serum. In addition, T cells were also shown to enhance the primary immune response to GXM. BALB/cBy mice were given GXM and anti-mouse thymocyte serum on day 1. On day 2, the experimental group was given anti-mouse thymocyte serum and the control group was given saline. On day 5, comparison of the PFC responses and anti-GXM antibody titers of the two groups revealed a significant increase in the immune response of the control over the experimental group. The type 2 T-cell-independent quality of GXM was also demonstrated in CBA/cHN xid mice. These mice lack the Lyb+ subset of B cells and are unable to respond to type 2 T-independent antigens but respond normally to type 1 T-independent antigens. Type III pneumococcal polysaccharide, a type 2 T-independent antigen, was used as a negative control, and trinitrophenyl-lipopolysaccharide, a type 1 T-independent antigen, was used as a positive control. The CBA/cHN xid mice failed to respond to either type III pneumococcal polysaccharide or GXM but did not respond to immunization with trinitrophenyl-lipopolysaccharide. BALB/cBy mice responded normally to all three antigens.     

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.     

T-cell-independent and T-cell-dependent B-cell responses to exposed variant surface glycoprotein epitopes in trypanosome-infected mice.

The T-cell dependency of B-cell responses to variant surface glycoprotein (VSG) epitopes exposed in their native surface conformation on Trypanosoma brucei rhodesiense clone LouTat 1 was investigated. T-cell requirements were examined by analyses of gamma globulin preparations derived from trypanosome-infected BALB/c nude (nu/nu) and thymus-intact (nu/+) mice. A radioimmunoassay was used to selectively quantitate antibody binding to native VSG 1 epitopes present on the surface of viable trypanosomes. Such analyses of VSG-specific antibody in infected mice demonstrated that in the absence of T cells there was a significant B-cell response to exposed VSG epitopes; however, in the presence of T cells these surface epitope-specific responses were greatly enhanced. In contrast to infection, immunization of mice with purified VSG 1 or paraformaldehyde-fixed parasites elicited significant VSG surface epitope-specific responses only in the presence of T cells (i.e., in nu/+ mice only). VSG-specific antibody responses in mice infected with three other clonal T. brucei rhodesiense populations (LouTat 1.2, 1.5, and 1.9) were found to be similar in this pattern, although not identical, to the anti-LouTat 1 responses. An important exception was that mice infected with LouTat 1.8 required T cells to produce VSG surface-specific antibody. Thus, the VSG surface epitope-specific B-cell responses in trypanosome-infected mice represent composite T-cell-independent and T-cell-dependent processes, and a significantly stronger response is made in the presence of T cells. However, immunization with VSG in the absence of infection elicited only T-cell-dependent responses. Since the relative contribution of T-cell-independent and T-cell-dependent processes to the total VSG-specific antibody produced during infection was variable (as seen with the absence of a T-cell-independent response to LouTat 1.8), this may reflect differences in the primary structure or display of VSG molecules on the trypanosome membrane or may represent active parasite interference with some epitope-specific B-cell responses.