Secondary Antibody Response Research Articles

Time Course and Metabolic Costs of a Humoral Immune Response in the Little Ringed Plover Charadrius dubius

Despite host defense against parasites and pathogens being considered a costly life-history trait, relatively few studies have assessed the energetic cost of immune responsiveness. Knowledge of such energetic costs may help to understand the mechanisms by which trade-offs with other demanding activities occur. The time course and associated metabolic costs of mounting a primary and secondary humoral immune response was examined in little ringed plovers Charadrius dubius challenged with sheep red blood cells. As was expected, the injection with this antigen increased the production of specific antibodies significantly, with peaks 6 d postinjection in both primary and secondary responses. At the peak of secondary antibody response, the antibody production was 29% higher than that observed during the primary response, but the difference was nonsignificant. Mounting the primary response did not significantly increase the resting metabolic rate (RMR) of birds, whereas the secondary response did by 21%, suggesting that the latter was more costly in terms of RMR. In spite of the fact that the primary response did not involve an increase in RMR, birds significantly decreased their body mass. This could imply an internal energy reallocation strategy to cope with the induced immune challenge. Last, we found that RMR and antibody production peaks were not coupled, which could help to conciliate the variable results of previous studies. Collectively, the results of this study support the hypothesis that humoral immunity, especially the secondary response, entails energetic costs that may trade-off with other physiological activities.

Effects of dietary selenium and vitamin E on immune response and biological blood parameters of broilers reared under thermoneutral or heat stress conditions

A study was conducted using 360 broiler chickens to evaluate the effects of dietary vitamin E (0, 125 and 250 mg/kg), selenium (Se, 0, 0.5 and 1 mg/kg), or their different combinations on immune response and blood biological parameters of broilers raised under either thermoneutral (TN, 23.9 °C constant) or heat stress (HS, 23.9 to 37 °C cycling) conditions. Humoral immunity was assessed by intravenous injection of 7% sheep red blood cell (SRBC) followed by evaluation of serum for antibody titers in primary and secondary responses. Heterophil to lymphocyte (H/L) ratio also determined as an indicator of stress. Furthermore, at the end of the experiment, birds were bled for determination of some biological parameters. There was a significant reduction in body weight and feed intake, but the feed conversion ratio increased when the birds were exposed to HS (P<0.05). Body weight and feed intake were not influenced significantly by dietary vitamin E and Se (P>0.05), whereas feed conversion was improved significantly by 125 mg/kg vitamin E (P<0.05). The liver and lymphoid organ weights as well as IgM and IgG, antibody titers for primary and secondary antibody responses to SRBC were reduced significantly under HS (P<0.05). Heat stress also resulted in a significant increase in H/L ratio (P<0.05). Dietary vitamin E resulted in improvement of primary and secondary antibody responses both in TN and HS broilers (P<0.05). The HS birds also showed an improved antibody titer in secondary response with high concentration of Se (P<0.05). Vitamin E and Se had interactive effects on anti-SRBC titers; however, no consistent differences were found between dietary levels during the study. The H/L ratio decreased by feeding vitamin E at both levels either under HS or TN conditions (P < 0.05). The serum concentrations of glucose, triglycerides, total cholesterol, and LDL-cholesterol were increased but serum HDL-cholesterol decreased in HS broilers (P<0.05).

Beraprost enhances production of antigen-specific IgG isotypes without modulating germinal center B cell generation and the affinity maturation

Immune regulating functions of lipid mediators are being recognized. Prostaglandins (PGs) are derived from phospholipid by cyclooxygenase-2 (COX-2) in inflammatory tissues. Based upon our previous data that imply an immune modulating activity of prostacyclin, we hypothesized that PGs promote the humoral immune responses in vivo. To test this hypothesis, we examined the effect of a prostacyclin analog beraprost and PGE2 on the antibody responses that were induced by immunizing mice with keyhole limpet hemocyanin (KLH). Beraprost was used due to the extremely unstable property of prostacyclin. Our results showed that beraprost indeed promoted the production of anti-KLH antibodies, which was dose-dependent and specific to beraprost because PGE2 did not modulate the antibody response compared with controls. The enhancing effect of beraprost was reproduced in the secondary responses, suggesting that memory B cell generation during the primary response was significantly affected by beraprost. Analysis of the isotypes of anti-KLH antibodies revealed that beraprost stimulated the production of IgA and IgG subisotypes but not IgM. However, germinal center B cell generation and the affinity of anti-KLH antibodies were not affected by beraprost administration. To confirm these results we immunized COX-2 knockout mice with KLH and analyzed whether the results with wild-type mice were reflected in the absence of PGs. The primary and secondary antibody responses were significantly impaired in the KO animals. The levels of anti-KLH IgG subisotypes and IgA were severely reduced in KO mice whereas those of IgM were comparable to controls. These results reveal an unrecognized function of PG in the humoral immune responses.

Investigating the Role for IL-21 in Rabies Virus Vaccine-induced Immunity

Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R−/−) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R−/− mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R−/− mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R−/− mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.

A Sub Acute Immunotoxicity Study in G?ttingen Minipigs? with the Immunosuppressive Compounds Cyclosporin A and Dexamethasone

Introduction: There is a growing interest in the minipig as a non-rodent species in the safety assessment of (bio) pharmaceuticals and (agro) chemicals. For (bio) pharmaceuticals the need to evaluate the potential adverse effects on the immune system is an increasingly important aspect of safety evaluation. In the present study, cyclosporin A and Dexamethasone were used as model compounds to examine whether the regulatory endpoints requested for immunotoxicity testing can be tested in the Gottingen minipig. Methods: Minipigs were treated with vehicle, 20 mg/kg/day cyclosporine A or 0.4 mg/kg/day dexamethasone for 39 (males) or 40 (females) consecutive days. Clinical signs, body weight, hematology, lymphocyte subset analysis in peripheral blood mononuclear cells, Natural Killer cell activity, primary and secondary antibody response and Delayed Type Hypersensitivity response against Keyhole Limpet Hemocyanin (KLH), ex vivo mitogen-induced lymphocyte proliferation, examination at necropsy for gross macroscopic changes, weights and histopathology of lymphoid organs were used as criteria for evaluation of immunotoxic effects. Results: Most parameters measured were implemented successfully in the minipig. Cyclosporin A treatment of minipigs had a slight effect on thymus weight, white blood cell counts and Keyhole Limpet Hemocyanin specific IgM responses. Clear effects were observed on mitogen-induced proliferation, Keyhole Limpet Hemocyanin specific IgG responses and Delayed Type Hypersensitivity response. Dexamethasone treatment resulted in decreased body weight, white blood cell counts, proliferative response to mitogen stimulation, Natural Killer cell activity and thymus weight. Keyhole Limpet Hemocyanin specific antibody responses were unaffected or slightly increased after dexamethasone treatment. Discussion: With few exceptions, results obtained with cyclosporine A and dexamethasone in the minipig was in accordance with those presented for other species in the literature. Overall, the results presented in this article indicate that the minipig has potential to serve as an alternative non-rodent species for immunotoxicity testing as part of the safety assessment of (bio) pharmaceuticals and chemicals.

VACCINATION of Atlantic salmon leads to long-lasting higher levels of serum immunoglobulin and possible skewed ratios of two distinct IgM isotypes

Primary and secondary antibody responses in blood of teleost fish are mainly IgM mediated, as they lack an IgG homolog and a class switch mechanism. Thus, the major serum immunoglobulin is tetrameric IgM. A unique antibody class in teleosts; named IgT, appears to be specialized for mucosal immunity and is present in low concentrations in serum. A third class; IgD was recently detected in serum of rainbow trout, but is less abundant than IgT. In the present study, relative quantification of total IgM showed that Atlantic salmon (Salmosalar) which had received an oil-adjuvanted commercial vaccine maintained about 2-fold higher levels of IgM; 14 and 17 months after intraperitoneal injection, in comparison to unvaccinated fish kept in the same cage. Similar results were obtained by ELISA (serum IgM) and reverse transcription real time PCR (spleen mRNA). Analyses conducted in our lab have shown that several salmonid species possess two slightly different IgM isotypes as a result of ancestral tetraploidy. In Atlantic salmon, two distinct IgM subpopulations (A and B) can be separated by anion exchange chromatography. The IgM-B tetramer exhibits a higher degree of disulfide cross-linking than IgM-A, presumably due to an extra cysteine near the C-terminus of the heavy chain. The typical A/B ratio is approximately 40/60. Anion exchange elution profiles of serum IgM from vaccinated fish indicate that prolonged triggering of the immune system can lead to a skewed ratio of IgM-A/IgM-B. In the context of recent results from rainbow trout (Onchorhynchusmykiss), showing that high affinity antibodies are more highly polymerized and have a longer half life time, Atlantic salmon is an interesting model to elucidate these aspects further since tools are available to distinguish IgM-A and IgM- B in this species.

Evaluation of canine T-cell dependent antibody response to the primary and secondary immunization with keyhole limpet hemocyanin

T-cell dependent antibody response (TDAR) incorporating both primary and secondary responses to keyhole limpet hemocyanin (KLH) in canine models have not yet been fully understood. To develop a practical dog TDAR model, we characterized primary and secondary antibody responses by intravenous or intramuscular immunization of KLH twice at intervals of 8 days during a 28-day course of study. Primary immunization with KLH by both routes induced a maximum IgM response on 6 to 8 days after the treatment, whereas the IgG response started 6 to 8 days after the treatment with relatively low levels. Remarkable increases in anti-KLH IgG levels (about 10-times compared with the primary response) were produced 5 to 7 days after the secondary KLH immunization by both routes. These results indicate that IgM-predominant and IgG-predominant responses were respectively induced by the primary and secondary immunization. Furthermore, the intravenous route showed higher baseline titers of primary and secondary anti-KLH IgM responses, suggesting that intravenous immunization of KLH might be a more suitable method for immunotoxicity evaluation. No remarkable inter-individual variability was noted in our canine models. Treatment with cyclophosphamide at 2 mg/kg/day for a consecutive 28 days significantly suppressed primary and secondary anti-KLH IgM and IgG responses induced by KLH injection on Days 15 and 23 of CPA treatment. These results demonstrate that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in dogs when exposed to potential immunomodulatory drugs.

Immunostimulatory effect of water soluble fraction ofNyctanthes arbortristisleaves on the immune response inOreochromis mossambicus(Peters)

Use of immunostimulants as a prophylactic measure against diseases in fish is considered as an effective alternative to antibiotic use. Plant-derived immunostimulants have recently received more attention, as most of them are cost-effective and eco-friendly. This study was carried out with the objective of evaluating the possible immunostimulatory activity of intraperitoneally injected water soluble fraction of Nyctanthes arbortristis (an Indian medicinal plant) leaves on (i) specific immunity (antibody response), (ii) nonspecific immunity (Lysozyme activity, ROS production) and (iii) functional immunity in terms of disease resistance against Aeromonas hydrophila in Oreochromis mossambicus. The results of the study showed that all the tested doses (i.e. 3.2, 16, 80 and 400 mg kg � 1 ) of water soluble fraction of Nyctanthes arbortristis leaves significantly enhanced both primary and secondary antibody responses to heat killed A. hydrophila. Significant enhancement in serum lysozyme activity by all the doses of water soluble fraction was observed on day 10. The lower doses of 3.2 and 16 mg kg � 1 of water soluble fraction alone enhanced intracellular ROS production. Fish treated with both single and double dose of water soluble fraction showed maximum disease resistance. The highest dose (400 mg kg � 1 ) of water soluble fraction exhibited highest relative per cent survival in both single dose and double dose groups.

Antibody repertoire development in fetal and neonatal piglets. XV. Porcine circovirus type 2 infection differentially affects serum IgG levels and antibodies to ORF2 in piglets free from other environmental factors

Porcine circovirus type 2 (PCV2) is an important pathogen in the porcine respiratory disease complex (PRDC) and its persistence may be due to dysregulation of systemic immunity. We examined this contention using isolator piglets. We present data on Ig levels in serum and bronchio-alveolar lavage (BAL), on antibody response to PCV2 and to TNP conjugates used as model antigens in 48 PCV2-infected isolator piglets. We compared these to data from TNP-immunized isolator piglets colonized with a probiotic flora, those infected with swine influenza (S-FLU) and those infected with porcine respiratory and reproductive syndrome virus (PRRSV). We found that PCV2 infection does not cause generalized hypergammaglobulinemia that characterizes PRRSV infections, but causes an unexplained increase in serum IgA. All animals had serum IgG to the ORF2 gene product of PCR2, but neither IgA nor IgG anti-ORF2 responses in BAL. PCV2 infection is a poor adjuvant since only natural anti-TNP antibodies were found. Unexpectedly, immunization appeared to result in lower Ig levels and lower anti-ORF2 responses. There was extreme variation in serum Ig levels in response to infection that could in part be traced to genetic and gender differences. These data suggest that non-replicating vaccines are unlikely to result in a significant primary antibody response but may prime the system for a secondary antibody and cytotoxic response following actual infection. In any case, developers may have to contend with significant genetic differences in the response of piglets to PCV2.

Ontogeny of Adaptive Antibody Response to a Model Antigen in Captive Altricial Zebra Finches

Based on studies from the poultry literature, all birds are hypothesized to require at least 4 weeks to develop circulating mature B-cell lineages that express functionally different immunoglobulin specificities. However, many altricial passerines fledge at adult size less than four weeks after the start of embryonic development, and therefore may experience a period of susceptibility during the nestling and post-fledging periods. We present the first study, to our knowledge, to detail the age-related changes in adaptive antibody response in an altricial passerine. Using repeated vaccinations with non-infectious keyhole limpet hemocyanin (KLH) antigen, we studied the ontogeny of specific adaptive immune response in altricial zebra finches Taeniopygia guttata. Nestling zebra finches were first injected at 7 days (7d), 14 days (14d), or 21 days post-hatch (21d) with KLH-adjuvant emulsions, and boosted 7 days later. Adults were vaccinated in the same manner. Induced KLH-specific IgY antibodies were measured using ELISA. Comparisons within age groups revealed no significant increase in KLH-specific antibody levels between vaccination and boost in 7d birds, yet significant increases between vaccination and boost were observed in 14d, 21d, and adult groups. There was no significant difference among age groups in KLH antibody response to priming vaccination, yet KLH antibody response post-boost significantly increased with age among groups. Post-boost antibody response in all nestling age groups was significantly lower than in adults, indicating that mature adult secondary antibody response level was not achieved in zebra finches prior to fledging (21 days post-hatch in zebra finches). Findings from this study contribute fundamental knowledge to the fields of developmental immunology and ecological immunology and strengthen the utility of zebra finches as a model organism for future studies of immune ontogeny.

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.

A β-mannan trisaccharide conjugate vaccine aids clearance of Candida albicans in immunocompromised rabbits

A β-(1→2)-linked mannose trisaccharide epitope that is the optimal inhibitor of two protective monoclonal antibodies specific for the Candida albicans cell wall phosphomannan was used to create a synthetic conjugate vaccine. Two injections of the trisaccharide-tetanus toxoid conjugate administered with alum induced a robust secondary antibody response in rabbits with trisaccharide specific IgG ELISA titers in excess of 1:100,000. Fluorescent labeling studies demonstrated these antibodies (i) recognized the cell wall β-mannan of C. albicans on hyphae and budding cells and (ii) C. albicans incubated with immune sera bound complement factor C3. The synthetic conjugate vaccine but not the carrier protein, tetanus toxoid reduced Candida load in vaccinated rabbits subsequently rendered leukocytopenic by injection of cyclophosphamide and then challenged with live C. albicans. These data support the contention that antibody mediated immunity plays a role in combating C. albicans infections and suggests that a surprisingly simple, readily accessible synthetic conjugate vaccine may have therapeutic potential.

Sleep and Antibody Response to Hepatitis B Vaccination

Experimental evidence links poor sleep with susceptibility to infectious illness; however, it remains to be determined if naturally occurring sleep is associated with immune responses known to play a role in protection against infection. The aim of this study was to determine whether sleep duration, sleep efficiency, and sleep quality, assessed in the natural environment, predict magnitude of antibody responses to a novel antigen among community volunteers in midlife. Observational. Healthy midlife adults (n = 125; 70 female; age 40-60 yr) received the standard 3-dose hepatitis B vaccination series. Actigraphy and electronic sleep diaries were used to assess sleep duration, sleep efficiency, and subjective sleep quality. Viral-specific antibody titers were obtained prior to the 2nd and 3rd vaccination to assess primary and secondary antibody responses. Clinical protection status (anti-hepatitis B surface antigen immunoglobulin G ≥ 10 mIU/ml) was assessed 6 mo after the final immunization. Regression analyses revealed that shorter actigraphy-based sleep duration was associated with a lower secondary antibody response independent of age, sex, body mass index, and response to the initial immunization. Shorter sleep duration, measured by actigraphy and sleep diary, also predicted a decreased likelihood of being clinically protected from hepatitis B at the conclusion of the vaccination series. Neither sleep efficiency nor subjective sleep quality were significant predictors of antibody response. Short sleep duration in the natural environment may negatively affect in vivo antibody responses to novel antigens, providing a possible explanation for observed associations of poor sleep with increased susceptibility to infectious disease.

Dietary selenium fortification: a potential solution to chronic arsenic toxicity

Selenium (Se), an important micronutrient and antioxidant, also acts as an antagonist of arsenic (As). Se supplementation of diet was investigated in mitigating chronic As toxicity in mammals. Experiments were conducted to determine whether Se supplementation in As-exposed rats might (i) decrease As-induced lipid peroxidation in liver, (ii) increase blood antioxidant status, and (iii) reverse suppression of the secondary antibody response. Male Wistar rats were exposed to As (40 and 80 ppm) in drinking water and received challenge diets with three different levels of Se (deficient: <0.01 ppm, adequate: 0.15 ppm, and fortified: 0.6 ppm) for 16 weeks. Clinical variables including behavior, body weight, and food and water consumption were recorded weekly, and blood sample was collected monthly. Antioxidant status was assessed through glutathione sulfhydryl (GSH) levels in whole blood. Lipid peroxidation in the liver was evaluated using the malondialdehyde (MDA) assay. The antibody response was measured using keyhole limpet hemocyanin as an immunogen. Se deficiency significantly increased hepatic lipid peroxidation and suppressed antibody production relative to the Se-adequate and -fortified groups, confirming that Se deficiency exacerbates the damage produced by As exposure. Se fortification markedly elevated the blood GSH level in both As-exposed groups, indicating protective effects. At adequate Se levels, rats showed signs of counteracting As-mediated toxicity. However, Se fortification produced more pronounced benefits against As-induced toxicity, a pattern that was particularly notable in the 40 ppm As group.

PRIMARY AND SECONDARY ANTIBODY RESPONSE TO COMPOSITE FORMALIN-INACTIVATED &lt;i&gt;STAPHYLOCOCCUS AUREUS&lt;/i&gt;, &lt;i&gt;STREPTOCOCCUS AGALACTIAE&lt;/i&gt; AND &lt;i&gt;ESCHERICHIA COLI&lt;/i&gt; IN RABBITS

Twenty buffaloes clinically positive for mastitis were selected for the collection of milk samples. Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli were isolated from milk of mastitic buffaloes according to the procedures recommended by National Mastitis Council Inc., USA. The composite antigen containing S. aureus, Str. Agalactiae and E. coli was then prepared and the concentration of each isolate was adjusted to 1 x 109 cells / ml. To evaluate the antibody response, nine adult healthy rabbits were divided randomly into three groups (A, B and C) consisting of 3 rabbits each. Composite antigen preparation was injected subcutaneously to the rabbits of groups A and B @ 0.2 ml / rabbit while each rabbit of group C inoculated with 0.2 ml normal saline and served as control. The rabbits of group B were given a booster dose at day 15 of the primary injection to see the secondary antibody response. The level of antibodies specific to S. aureus, Str. agalactiae and E. coli were assayed by indirect haemagglutination test (HA) and finally geometric mean titre (GMT) was calculated. At day 0 to 15, it was observed that the antibody titre was almost same in both groups. At day 30 significant difference in antibody titres was observed between group A and B with GMT of 9.8 against S. aureus, and Str. agalactiae in group A whereas in group B GMT was 97.0 against S. aureus and 39.4 against Str. agalactiae which was higher than group A. The GMT against E. coli was 12.1 and 24.3 in group A and B, respectively. At day 45 and 60 there was progressive decrease in antibody titre against Str. agalactiae in group B. The antibody titre against S. aureus first increased at day 45 and then decreased at day 60 whereas the antibody titre against E. coli remained persistent till day 60 in rabbits of group B. Furthermore, the primary antibody response to E. coli of composite antigen was higher (GMT, 12.1 at day 15) compared with S. aureus and Str. agalactiae (GMT, 9.8 at day 15) whereas the secondary antibody response to S. aureus was higher (GMT, 128) compared with Str. agaiactiae and E. coli (GMT, 24.3 at day 45).

The immunogenic characteristics associated with multivalent display of Vi polysaccharide antigen using biodegradable polymer particles

Polysaccharides in their great majority are thymus-independent (TI) antigens. Anti-polysaccharide antibody responses are generally weak and characterized by lack of memory, isotype restriction and delayed ontogeny. We report here the generation of protective memory antibody response by multivalent display of polysaccharide antigens on biodegradable polymeric particles. Single dose immunization using polylactide (PLA) polymer particles entrapping Vi capsular polysaccharide antigen from Salmonella typhi promoted isotype switching and induced polysaccharide-specific memory antibody response in experimental animals. PLA nanoparticles as well as microparticles entrapping Vi polysaccharides elicited high IgG titer in comparison to soluble Vi immunization. Immunizations with particles co-entrapping both Vi polysaccharide and tetanus toxoid did not improve the anti-polysaccharide antibody responses. Lower antibody response from co-entrapped formulation was mostly due to inhibition of particle phagocytosis by the macrophages. Immunization using polylactide particles entrapping only Vi polysaccharide with higher density on surface elicited highest secondary antibody response as well as promoted isotype switching. The vaccination potential of particle based immunizations was further confirmed by the generation of quick memory antibody responses while challenging the immunized animals with live S. typhi. This approach provides a multivalent display of polysaccharide antigen using polymer particles and elicits protective memory antibody response without conjugation to a carrier protein

T cell independent secondary antibody responses to the envelope protein of simian immunodeficiency virus

BackgroundDuring human (HIV) and simian (SIV) immunodeficiency virus infection, loss of CD4+ T cells and progression to AIDS are associated with a decline in antibody titers to the viral Gag protein, while antibodies to the Env protein remain high, suggesting a T cell independent antibody response to Env.ResultsTo explore differential regulation of Gag and Env antibody responses, immunocompetent BALB/c and T cell deficient nude mice were immunized with virus like particles (VLP) of simian immunodeficiency virus or adenoviral vectors expressing SIV Gag and Env. High levels of antibodies against Gag and Env could only be induced in immunocompetent mice, but not in the immunodeficient mice. Thus, neither cells expressing Env after adenoviral gene transfer nor VLPs induce a T cell independent primary anti-Env antibody response. However, secondary B cell responses to Env, but not to Gag, were observed in immunodeficient mice after transfer of primed B cells and boosting with VLPs or adenoviral vectors expressing Gag and Env. This T cell independent secondary antibody response to Env was reduced after stimulation with VLPs modified to contain monomeric membrane bound gp130 surface subunit of Env and undetectable after injection of soluble gp130.ConclusionsMembrane-bound trimeric Env seems to be responsible for the maintenance of high levels of anti-Env antibodies during progression to AIDS. This T cell independent secondary antibody response may prevent T cell-dependent affinity maturation and thus contribute to viral immune escape by favoring persistence of non-protective antibodies.

Differential contribution of dendritic cell CD1d to NKT cell-enhanced humoral immunity and CD8+ T cell activation

CD1d-restricted type I NKT cells provide help for specific antibody production. B cells, which have captured and presented a T-dependent, antigen-derived peptide on MHC class II and CD1d-binding glycolipid α-GC on CD1d, respectively, activate Th and NKT cells to elicit B cell help. However, the role of the DC CD1d in humoral immunity remains unknown. We therefore constructed mixed bone marrow chimeras containing CD1d-expressing, DTR-transgenic DCs and CD1d(+) or CD1d(-) nontransgenic DCs. Following DT-mediated DC ablation and immunization, we observed that the primary and secondary antibody responses were equivalent in the presence of CD1d(+) and CD1d(-) DCs. In contrast, a total ablation of DCs delayed the primary antibody response. Further experiments revealed that depletion of CD1d(+) DCs blocked in vivo expansion of antigen-specific cytotoxic (CD8(+)) T lymphocytes. These results provide a clear demonstration that although CD1d expression on DCs is essential for NKT-enhanced CD8(+) T cell expansion, it is dispensable for specific antibody production.

Antigen-specific antibody responses in B-1a and their relationship to natural immunity

B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3-5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.