Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems)

The application of LIPS to the rapid quantification of antibody responses to infectious agents is described. Chimeric genes encoding pathogen antigens fused to Renilla luciferase are expressed in mammalian cells; crude extracts are prepared and, without purification, employed in immunoprecipitation assays to quantify pathogen-specific antibodies. In cross-sectional and longitudinal studies, antibody levels to the MSG-14 antigen of Pneumocystis jirovecii measured by this assay correlated well with levels previously obtained with an optimized ELISA. We also correctly predicted Hepatitis B (HBV), Hepatitis C (HCV), and HIV infection status in all but 2 of 99 assays analyzing 33 patient sera. We then used 15 HIV-encoded proteins comprising the whole HIV proteome to generate antibody response profiles for these 33 sera. Each HIV antigen was recognized by antibodies in serum from at least one HIV-infected individual. Data generated with these simple, quantitative antibody-detection assays have both clinical and research applications.     

Use of tetrameric antibody complexes to stain cells for flow cytometry

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.     

Excretion of human immunodeficiency virus type 1 through polarized epithelium by immunoglobulin A

Human immunodeficiency virus (HIV) is transmitted primarily sexually across mucosal surfaces. After infection, HIV propagates initially in the lamina propria below the polarized epithelium and causes extensive destruction of mucosal T cells. Immunoglobulin A (IgA) antibodies, produced in the lamina propria and then transcytosed across the mucosal epithelium into the lumen, can be the first line of immune defense against HIV. Here, we used IgA monoclonal antibodies against HIV envelope proteins to investigate the abilities of polarized primate and human epithelial cells to excrete HIV virions from the basolateral to the apical surface via polymeric Ig receptor (pIgR)-mediated binding and the internalization of HIV-IgA immune complexes. African green monkey kidney cells expressing pIgR demonstrated HIV excretion that was dependent on the IgA concentration and the exposure time. Matched IgG antibodies with the same variable regions as the IgA antibodies and IgA antibodies to non-HIV antigens had no HIV excretory function. A mixture of two IgA anti-bodies against gp120 and gp41 showed a synergistic increase in the level of HIV excreted. The capacity for HIV excretion correlated with the ability of IgA antibodies to bind HIV and of the resulting immune complexes to bind pIgR. Consistent with the epithelial transcytosis of HIV-IgA immune complexes, the colocalization of HIV proteins and HIV-specific IgA was detected intracellularly by confocal microscopy. Our results suggest the potential of IgA antibodies to excrete HIV from mucosal lamina propria, thereby decreasing the viral burden, access to susceptible cells, and the chronic activation of the immune system.     

Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses.

We developed an in vitro assay for antibodies to hepatitis C virus (HCV) that bind to virions and prevent initiation of the replication cycle in susceptible cells in vitro. These antibodies therefore appear to be capable of neutralizing the virus. Using this assay and a standard inoculum of HCV of known infectivity, we have measured the antibody in serial serum samples obtained from the same chronically infected patient over 14 years following onset of his hepatitis. Such antibody was found in sera collected within 5 years of onset of hepatitis but not in later sera. In double immunoprecipitation experiments with anti-human immunoglobulin, the same sera that contained neutralizing antibody were found to contain antibody that bound to HCV to form antigen-antibody complexes immunoprecipitable with anti-human globulin. Similarly, plasma collected from this patient in 1990, 13 years after onset of hepatitis, and which contained HCV that had diverged genetically from the 1977 strain, did not contain antibody capable of neutralizing either the 1977 or the 1990 strain of HCV. However, plasma collected a year later (1991, 14 years after onset of hepatitis) contained neutralizing antibody to the 1990, but not the 1977, strain of HCV. These results suggest that HCV does induce antivirion antibody, as measured by blocking of initiation of the replication cycle of virus in cells and by the formation of immunoprecipitable antigen-antibody complexes but that these antibodies are isolate specific and change over time. Thus, these antivirion antibodies function as neutralizing antibodies and are probably in vitro correlates of the attempt of the host to contain the emergence of neutralization-resistant variants of HCV over time.     

Antibody-dependent enhancement of hepatitis C virus infection

Hepatitis C virus (HCV) often causes a persistent infection associated with hypergammaglobulinemia, high levels of antiviral antibody and circulating immune complexes, and immune complex disease. We previously reported that only a limited neutralizing activity to vesicular stomatitis virus or HCV pseudotype is generated in animals immunized with recombinant HCV envelope proteins and chronically infected HCV patient sera. Interestingly, when some of these neutralizing sera were diluted into a range of concentrations below those that reduced virus plaque number, an increase in pseudotype plaque formation was observed. Purified HCV E2-specific human monoclonal antibodies were used to further verify the specificity of this enhancement, and one- to twofold increases were apparent on permissive Huh-7 cells. The enhancement of HCV pseudotype titer could be inhibited by the addition of a Fc-specific anti-human immunoglobulin G Fab fragment to the virus-antibody mixture prior to infection. Treatment of cells with antibody to Fc receptor I (FcRI) or FcRII, but not FcRIII, also led to an inhibition of pseudotype titer enhancement in an additive manner. Human lymphoblastoid cell line (Raji), a poor host for HCV pseudotype infection, exhibited a four- to sixfold enhancement of pseudotype-mediated cell death upon incubation with antibody at nonneutralizing concentrations. A similar enhancement of cell culture-grown HCV infectivity by a human monoclonal antibody was also observed. Taken together, antibodies to viral epitopes enhancing HCV infection need to be taken into consideration for pathogenesis and in the development of an effective vaccine.     

Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes

We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.     

Activation of T Cells by Autoantigen Immobilized by Specific Antibodies

A convenient microtiter-plate assay that uses immobilized antibody to capture specific antigens for presentation to T cells has been developed. Initial experiments used KLH as the antigen, immune antisera and draining lymph node cells from immunized NOD mice as the source of antibody and T cells, and spleen cells from naive NOD mice as the source of antigen-presenting cells (APCs). The resulting proliferation of the T cells was shown to be antibody- and antigen-specific, suggesting that the APCs had internalized and processed the captured antigen, presenting it to the T cells in the form of peptide/MHC complexes. The approach was also tested for an autoimmune disease as part of an effort to identify autoantigens responsible for the proliferation of T cells in the synovial fluid of rheumatoid arthritis patients. When immunoglobulin from autologous synovial fluid was captured on plates coated with anti-human immunoglobulin antibodies, the addition of HLA-DR4 peripheral blood mononuclear cells as APCs and synovial fluid-reactive HLA-DR4-restricted T-cell clones resulted in significant proliferation, indicating that the specific antigen in the crude synovial fluid was human immunoglobulin. This response was also shown to be antigen-specific and HLA-DR4-restricted. This assay format should permit the definition of autoantigens by capturing with antibodies to crude autoantigen extracts, followed by the addition of the appropriate APC and T-cell populations.     

In vitro immune response by murine bone marrow cells stimulated against soluble immune complexes.

Synchronized nonadherent bone marrow lymphocytes were stimulated with soluble immune complexes, in antigen excess formed by C3H/HeJ antibodies and various noncross-reacting protein antigens, in a suspension culture which allowed longterm cultivation. On binding of these complexes, lymphocytes underwent blast transformation with mitosis and formation of plasma cells which secreted specific antibodies to the antigen; a cyclic sequence of lymphocytes, blasts, and plasma cells was observed until the majority of the cell population appeared to be plasma cells. The relative percentage of mature plasma cells then decreased leaving mostly small lymphoid cells among which evidence suggests the presence of memory cells. Complexes at equivalence stimulated for the first few days, whereas antibody excess caused stimulation only initially followed by inhibition of the response. Antibodies passively added to the cultures inhibited the proliferative reaction; free antigen induced a typical secondary-type response.     

Antigen Binding to Secretory Immunoglobulin A Results in Decreased Sensitivity to Intestinal Proteases and Increased Binding to Cellular Fc Receptors

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA·antigen immune complexes are selectively transported across Peyer''s patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (FcαRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.     

同时检测血清中多种抗体的蛋白质微阵列研究

建立一种用蛋白质微阵列法可同时检测血清中艾滋病毒（HIV）, 丙型肝炎病毒（HCV）和梅毒螺旋体（TP）抗体的方法.将基因工程HIV、HCV和TP 3种融合抗原共价结合于固相载体玻片上,制成蛋白质微阵列,血清样本经稀释、加样、孵育、洗涤后,加上Cy3荧光标记二抗,洗涤,激光共聚扫描,将获得的图片用Bio-discover公司的Imagene专用分析软件进行分析,所获数据在经过自行编写的软件,根据Cutoff值自动生成判断结果.用此蛋白质微阵列系统检测了400例阴性血清,确定了Cutoff值,检测中国药品生物制品检定研究所的HIV, HCV和TP 3种参比品,并与3种ELISA试剂盒的结果进行了比较.蛋白质微阵列的艾滋病毒阳性和阴性符合率均为100%(20/20); 丙型肝炎病毒阳性和阴性符合率均为95%(38/40).梅毒螺旋体参比品阳性符合率为100%(10/10),阴性符合率为100%(20/20),蛋白质微阵列与三种ELISA试剂检测国家HIV、TP、HCV参比品（共150份血清样本）,结果具有高度的符合率.     

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.     

Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode

The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritin-conjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretion along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior.     

Studies on the role of neutralizing antibodies against envelope genes in resolving HCV pseudo-particles infection

Characterization of antibodies targeting the attachment and entry of the viral particles into host cells is important for studding antibody mediated neutralization. Antibodies against the envelope glycoproteins (EGP) have neutralizing capacity and can prevent HCV infections. System based on HCV pseudo typed-particles (HCVpp) stably expressing EGP can be used for screening of HCV anti envelope neutralizing antibodies in the serum of patients with acute and chronic HCV infections. The aim of the current study was to check HCVpp as a useful tool for the detection of anti-HCV envelope antibodies in the serum of HCV infected patients and to test the binding potential of these antiviral molecules to EGP of HCV 3a. Previously developed HCVpp harboring unmodified glycoproteins from local isolates in 293T cell line were used in this study. HCVpp were pre incubated with different concentrations of anti E1 antibody and different E2 antibodies to check antiviral activity. Further we used serum samples with low/medium (≤800,000 IU/mL), and high (>800,000 IU/mL) viral titer from chronic HCV male and female patients. Infection was done in Huh-7 cells for 1 h at 37 oC. Infectivity was checked through Luciferase assay. Considerable decrease in HCVpp infectivity with anti-envelope antibodies was observed in dose dependent manner. Maximum inhibition was seen when 5 µg/ml of monoclonal anti E1 antibody used. Further increase in concentration exhibited no decrease in infectivity which suggests that other factors are also involved in causing infection. Various well characterized E2-specific monoclonal antibodies (mAbs) have been screened for their capability to reduce infection in Huh-7 cells. Three of the four mAbs specific for the E2 had no effect on the infectivity of HCVpp. Confirmation sensitive antibody H53 showed maximum inhibition of infectivity. HCV ELISA positive samples from both male and female patients were used to neutralize the HCVpp. The neutralizing antibody response was observed in both males and females patients and do not assemble the rapidly evolving HCV envelope glycoproteins. That is why in spite the presence of neutralizing antibodies in the blood they fail to resolve infections. Moreover E1 antibodies insignificantly (>0.05) inhibit HCVpp infectivity while E2 antibodies significantly (<0.05) inhibit HCVpp infection. Based on the results of this study it is concluded that anti-envelope antibodies particularly the anti-E2 could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential for developing passive and active immunization for hepatitis C along with interferon therapy.     

Temporal relation of antigenaemia and loss of antibodies to core antigens to development of clinical disease in HIV infection.

A total of 276 sequential serum samples from 34 men with antibodies to the human immunodeficiency virus (HIV) followed up for two to seven years were analysed for HIV antigen and antibodies to the viral core and envelope proteins. Results were correlated with clinical outcome and CD4 T lymphocyte count. Both antigenaemia and the disappearance of antibodies to the core protein were associated with development of the acquired immune deficiency syndrome (AIDS) or AIDS related complex and depletion of CD4 cells. Thus AIDS or AIDS related complex developed in eight out of 16 patients with antigenaemia compared with one out of 18 patients without antigenaemia. Low counts of CD4 cells (less than 0.5 X 10(9)/l) were found in 14 of the 16 patients with antigenaemia and five of the 18 without antigenaemia. Nine patients seroconverted to HIV during the study; two of these developed antigenaemia 14 and 16 months after the estimated time of seroconversion. These results show that the late stages of HIV infection are characterised by increased production of antigen and a decrease in antibodies directed against the core protein. Antigenaemia indicates a poor prognosis; and as the antigen test is simple to do and interpret, it may therefore be useful for selecting patients for antiviral treatment.     

Antibody-mediated cell cytotoxicity in a defined system: regulation by antigen, antibody, and complement.

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.     

Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund’s adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8⁺ T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D^b-restricted and three HLA-A^*0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use.     

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8⁺ T cell responses. Moreover, E2-E1-LNP induced CD8⁺ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.     

Effective binding to protein antigens by antibodies from antibody libraries designed with enhanced protein recognition propensities

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.     

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role.     

Effects of carrageenan on immune responses. Studies on the macrophage dependency of various antigens after treatment with carrageenan.

Carrageenan, a sulfated polygalactose having macrophage toxic properties, elicited a marked suppression of IgM response to T cell-dependent antigens such as sheep red blood cells (SRBC), dinitrophenylated bovine serum gamma-globulin (DNP-BGG), and trinitrophenylated concanavalin A (TNP-Con A). In contrast, carrageenan did not inhibit antibody responses to such T cell-independent antigens as trinitrophenylated DEAE-dextran (TNP-DEAE-dextran), trinitrophenylated polyvinyl pyrrolidone(TNP-PVP), and trinitrophenylated Ficoll (TNP-Ficoll). Compared to total spleen cells, spleen cells from which macrophages had been removed by adhesion to plastic Petri dishes had less effect on the production of antibody against T cell-dependent antigens, but no change or a rather stimulated effect was observed in in vitro antibiody synthesis against T cell-independent antigens. These results strongly suggest that macrophages are involved in antibody responses to T cell-dependent antigens but not in those to T cell-independent antigens. However, the antibody response to trinitrophenylated lipopolysaccharide (TNP-LPS), a T cell-independent antigen, was inhibited by carrageenan treatment, suggesting that the response is macrophage dependent. Moreover, antibody response to higher doses of dinitrophenylated phytohemagglutinin (DNP-PHA), a T cell-dependent antigen, was shown to be macrophage independent by carrageenan treatment, although the antibody response to low doses of the antigen was macrophage dependent. Considering all these results, carrageenan treatment seems to be a very useful method to determine whether immune response to various antigens are macrophage dependent or not.