Immunization of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells

Fucosyl-GM1 (Fuc-GM1) [Fucα1 → 2Galβ1 → 3GalNAcβ1 → 4(NeuAcα2-3)Galβ1 → 4Glcβ1 → O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC. Received: 25 March 1999 / Accepted: 5 August 1999     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen-I (OFA-I)

Summary IgG anti-OFA-I found in melanoma patients was tested for its ability to lyse human tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Sera from 89 stage II melanoma patients which contained non-HLA-related IgG antibody to an OFA-I-positive melanoma cell line (M14) as tested by indirect membrane immunofluorescence (IMI) were originally chosen as possible sources of IgG anti-OFA-I. Of those tested for specific IgG activity to OFA-I by IMI, anti-OFA-I was found only in those patients immunized with OFA-I-positive tumor cells. When the same sera were tested in ADCC, no non-HLA-related activity could be demonstrated. This result was confirmed with purified IgG fractions that could, nevertheless, show anti-OFA-I reactivity in a complement-dependent cytotoxicity assay. The fact that naturally occurring IgG anti-OFA-I antibody was not readily detectable in patients' sera and that induced IgG anti-OFA-I did not participate in ADCC indicates that OFA-I-related tumor cell lysis via ADCC is an unlikely phenomenon in cancer patients.     

Carbohydrate antigens as targets for active specific immunotherapy

 Carbohydrate antigens such as GM2, GD2 and GD3 (gangliosides), Lewis^y and globo-H (neutral glycolipids and glycoproteins), and Tn, TF and sTn (glycoproteins) are overexpressed in a variety of cancers. Antibodies against several of these carbohydrate antigens have been detected in sera from patients treated with cancer vaccines, and have been associated with a more favorable prognosis. Clinical responses have been reported after treatment with monoclonal antibodies against some of these antigens. Hence cell-surface carbohydrate antigens have been identified as suitable targets for immune attack by both active and passive immunotherapies. Different approaches have been adopted to induce immune responses against these carbohydrate antigens. These includes vaccination with whole or lysed tumor cells, purified or synthetic carbohydrates, immunogenic carbohydrate derivatives, or carbohydrates conjugated with immunogenic carriers and administered with immunological adjuvants. In the case of gangliosides, immunization with either whole tumor cells or cell lysates has only occasionally induced responses against carbohydrate antigens, and the antibodies were generally IgM antibodies of low titer. Compared with other methods of vaccination, conjugate vaccines have consistently induced the highest titer of IgM and IgG antibodies against gangliosides and other carbohydrate antigens. Preclinical and clinical studies with conjugate carbohydrate vaccines have induced IgM and IgG antibody responses capable of inducing complement-mediated cytotoxicity of tumor cells in vitro and associated with prolonged disease-free and overall survival in patients. Received: 6 August 1996 / Accepted: 20 September 1996     

Analysis of effector cells in human antibody-dependent cellular cytotoxicity with murine monoclonal antibodies

We examined purified human large granular lymphocytes, peripheral monocytes, and T cells for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) with murine monoclonal antibodies. We also evaluated the effects of pretreatment of cells with interleukin 2 and interferon to augment ADCC activity. MB3.6, a murine monoclonal antibody directed against the GD3 ganglioside, induced high levels of ADCC. This ADCC was mediated predominantly, if not completely, by human killer cells (large granular lymphocytes) whereas other effector cell populations demonstrated no significant cytotoxic activity in 6- or 18-hr assays. The IgG2a an anti-melanoma antibody 9.2.27 generated low or no ADCC with most normal donors or melanoma patients. IL 2 was a very potent booster of ADCC activity. Interferon alpha also was effective, whereas interferon gamma did not augment but rather inhibited reactivity. We tested a large panel of antibodies of various isotype against colon carcinoma cells and found that gamma-3 isotype antibodies more frequently generated ADCC and produced higher levels of cytotoxic activity than did IgG1 or IgG2 antibodies. It appears that a variety of parameters can affect ADCC reactions, including the type of effector cell and its level of activation, the isotype of the antibody, and properties of the target cell line such as its susceptibility to lysis.     

Vaccination of Stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

Influence of adjuvants in inducing immune responses to different epitopes included in a multiepitope,multivalent, multistage Plasmodium falciparum candidate vaccine (FALVAC-1) in outbred mice

FALVAC-1, a vaccine against Plasmodium falciparum was developed by joining 21 epitopes from P. falciparum vaccine antigens and an universal T helper epitope from tetanus toxoid. Since adjuvants influence different aspects of immune responses, in this study we investigated the effect of four adjuvants aluminum hydroxide (alum), nonionic copolymer adjuvant P1005 (water-in-oil emulsion), CpG oligodeoxynucleotides (ODN), and QS-21 in eliciting immune responses in outbred mice. QS-21 and copolymer adjuvants were the best formulations in inducing higher and long-lasting antibody titers to the whole vaccine compared to alum and CpG. QS-21 was the only adjuvant to elicit predominantly IgG2a response and antibodies reactive with all epitopes incorporated in the vaccine construct. Vaccine elicited antibodies recognized sporozoites and asexual blood-stage parasites. FALVAC-1 immunized mice induced lymphoproliferative and IFN-gamma response to the vaccine. QS-21 and CpG adjuvants were able to elicit T proliferative responses to 20 of the 22 epitopes in the vaccine. In conclusion, this study demonstrated that with suitable adjuvant such as QS-21, it is possible to elicit immune responses to most of the epitopes included in the FALVAC-1 vaccine.     

A polyvalent vaccine for high-risk prostate patients: “are more antigens better?”

We have shown the immunogenicity and safety of synthetic carbohydrate vaccines when conjugated to the carrier keyhole limpet hemocyanin (KLH) and given with the adjuvant, QS-21, in patients with biochemically relapsed prostate cancer. To determine whether immune response could be further enhanced with stimulation by multiple antigens, a hexavalent vaccine was prepared using previously determined doses and administered in a Phase II setting to 30 high-risk patients. The hexavalent vaccine included GM2, Globo H, Lewis^y, glycosylated MUC-1-32mer and Tn and TF in a clustered formation, conjugated to KLH and mixed with QS-21. Eight vaccinations were administered over 13 months. All 30 patients had significant elevations in antibody titers to at least two of the six antigens; 22 patients had increased reactivity with FACS. These serologic responses were lower than that seen previously in patients treated with the respective monovalent vaccines. The reciprocal median combined IgM and IgG antibody titers with ELISA against MUC1, Tn, TF, globo H and GM2 for these 30 patients were 640, 80, 120, 40 and 0, compared to 1280, 640, 1280, 320 and 160 seen in patients receiving individual monovalent vaccines. This hexavalent vaccine of synthetic “self” antigens broke immunologic tolerance against two or more antigens in all 30 vaccinated patients, was safe, but antibody titers against several of the antigens were lower than those seen in individual monovalent trials. No impact on PSA slope was detected. We address the relevance of the multivalent approach for prostate cancer treatment. Supported by the Prostate Cancer Foundation, The PepsiCo Foundation, The Sharon Hels and Brad Reed Fund, Swim Across America, The Sara Chait Foundation. Dr. Philip Livingston is a consultant for and shareholder in Progenics Pharmaceuticals, Inc.     

Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients

Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without adjuvant, TF-KLH plus the immunological adjuvant Detox, sTn-KLH plus Detox, or sTn-KLH plus the immunological adjuvant QS-21, and the serological response was monitored. Enzyme-linked immunosorbent assay (ELISA), do-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus immunological adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins.This work was supported by grants from the National Institutes of Health (CA33049, CA52491, CA08748) and the Chemotherapy Foundation     

Monoclonal anti-human tumor antibodies of six isotypes in cytotoxic reactions with human and murine effector cells

Eighty-seven murine monoclonal antibodies (MAb) produced against human tumors of various origins and representing six different immunoglobulin classes were tested for antitumor reactivity in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. Mouse splenocytes, thioglycolate-elicited mouse peritoneal macrophages, freshly obtained nonadherent human peripheral blood lymphocytes, and human monocytes were used as effector cells, and human or rabbit serum as the source of complement. Of all four effector cell types tested, mouse macrophages showed the highest cytotoxic activity, based on net cytotoxicity, minimum requirement for Mab concentration, and effector cell number. Different immunoglobulin classes were associated with characteristic patterns of reactivity with the various effector cells or complement, independent of the target cell type used. MAb able to mediate ADCC were found among all IgG subclasses, with IgG2a and IgG3 MAb inducing lysis with all effector cell types. IgM and IgA MAb were nonreactive in the various ADCC assays, but IgM MAb were highly cytotoxic with complement.     

Carbohydrate vaccines that induce antibodies against cancer. 2. Previous experience and future plans

In conclusion  The primary function of antibodies is the elimination of circulating viral or bacterial pathogens from the blood-stream, lymphatics and interstitial spaces, and so, once induced, antibodies should be ideally suited for eliminating tumor cells and micrometastases from these spaces as well. Natural or tumor-induced and vaccine-induced antibodies against human cancer-associated antigens have been correlated with an improved clinical outcome. In the mouse, passive administration of monoclonal antibodies against cell-surface antigens 1–4 days after tumor challenge, and active induction of antibodies with vaccines, has resulted in prolonged survival or complete protection from tumor growth. This is a setting similar to the adjuvant setting in humans. Carbohydrates are the most abundant antigens at the cell surface of cancer cells, where they play important roles in cell-cell interactions, proliferation and the metastatic process. They have been shown to be excellent targets for immune attack by antibodies against human cancers, especially in the adjuvant setting. Vaccines containing these carbohydrate antigens covalently attached to immunogenic carrier proteins, such as KLH, plus potent immunological adjuvants, such as QS-21, effectively induce antibodies against these antigens in patients, which can result in complement-mediated lysis of antigen-positive tumor cells. Phase III trials with KLH conjugate vaccines have been initiated in the adjuvant setting against two carbohydrate antigens, the ganglioside GM2 and the blood-group-related antigen sTn. As the immunogenicity of additional vaccines is confirmed in small pilot trials, trials with polyvalent vaccines against two to five different antigens tailored for particular cancer types are planned.     

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers

Previously using a series of monovalent vaccines, we demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. We have prepared a heptavalent-KLH conjugate vaccine containing the seven epithelial cancer antigens GM2, Globo H, Lewis(y), TF(c), Tn(c), STn(c), and glycosylated MUC1. In preparation for testing this vaccine in the clinic, we tested the impact on antibody induction of administering the individual conjugates plus adjuvant compared with a mixture of the seven conjugates plus adjuvant, and of several variables thought to augment immunogenicity. These include approaches for decreasing suppressor cell activity or increasing helper T-lymphocyte activity (low dose cyclophosphamide or anti-CTLA-4 MAb), different saponin adjuvants at various doses (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). We find that: (1). Immunization with the heptavalent-KLH conjugate plus GPI-0100 vaccine induces antibodies against the seven antigens of comparable titer to those induced by the individual-KLH conjugate vaccines, high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10240), STn (640/5120), TF (320/10240), MUC1 (80/20480), and globo H (640/40); while lower titers of antibodies against Lewis(y)()(160/0) and only occasional antibodies against GM2 are induced. (2). These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. (3). None of the approaches for further altering the suppressor cell/helper T-cell balance nor changes to the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. (4). An optimal dose of saponin adjuvant, QS-21 (50 microg) or GPI-0100 (1000 microg), is required for optimal antibody titers. This heptavalent vaccine is sufficiently optimized for testing in the clinic.     

High-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells

Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses.     

The characterization of antibacterial antibodies in bovine immune sera to Staphylococcus aureus

Humoral antibody responses to the encapsulated Smith diffuse strain of Staphylococcus aureus were examined in cows immunized with the killed vaccine via different systemic routes. The sequential appearance of the antibody within different immunoglobulin classes in the sera during the course of immunization was followed by passive hemagglutination (PHA) and precipitation (PC) reactions and the mouse passive protection test. Repeated intravenous injections with the killed vaccine suspended in buffered saline stimulated production of IgM antibody exclusively during the whole period of immunization. On the contrary, following intramuscular administration with the vaccine incorporated in Freund's incomplete adjuvant, the antibodies appeared predominantly in IgG fractions of the sera. Specific antibody to the homologous strain used for vaccination was prepared from bovine immune sera by an absorption and elution process. The mouse passive protective activity of the antibody preparation was removed by absorption with the capsular polysaccharide antigen as well as by the whole cell adsorbent of the Smith diffuse strain, but not by the Smith compact and Cowan I strains of S. aureus. IgM, IgG1 and IgG2 proteins were isolated from the purified antibody and were compared, on a weight basis, with respect to their biological activities. Slightly higher activity of the IgG over the IgM antibody was demonstrated both in the mouse passive protection test and PC reaction, whereas in the PHA reaction, IgM antibody was shown to possess a significantly higher activity than IgG antibody. These studies suggest that IgG as well as IgM antibody might play an important role in protection against infection with encapsulated strains of S. aureus in cows.     

Protection against rotavirus-induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies.

Newborn mice suckled on dams immunized either orally or parenterally with primate rotavirus SA-11 were protected against diarrhea induced by SA-11 virus challenge. Experimental oral administration of milk from orally immunized dams protected suckling mice against challenge; protective activity was detected both in the anti-rotavirus immunoglobulin A (IgA) and IgG fractions, but IgA was more potent in vivo than IgG. Oral administration of milk from parentally immunized dams also protected suckling mice against challenge; in this case, protective activity was detected in the anti-rotavirus IgG fraction. In newborn mice foster-nursed by seronegative dams, circulating rotavirus-specific antibodies in high titer did not protect mice against oral SA-11 virus challenge. It appears that the most effective rotavirus vaccine will be that which induces an efficient production of antibodies active at the intestinal cell surface.     

Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies.

Antibodies that lyse trypomastigotes in a complement-mediated reaction are believed to be the main participants in the protection against virulent Trypanosoma cruzi. Antibodies with a specificity for alpha-galactosyl-containing determinants--generally called antiGal--were studied to determine their role in the lysis of trypomastigote forms. The titers of antiGal markedly increase in Chagas's disease. In the present study we demonstrate binding of this antibody to T. cruzi and the complement-mediated lysis of trypomastigotes by antiGal. Lysis of metacyclic trypomastigotes by whole Chagasic (Ch) serum or isolated antiGal fractions was equally inhibited by alpha- but not by beta-galactosides. Most of the lytic power of the Ch antiGal as well as of the whole Ch serum was removed by absorption on Synsorb-linked Gal alpha 1, 3Gal beta 1, 4GlcNAc followed by rabbit erythrocyte absorption. The Ch antiGal had a lower affinity for melibiose bound to agarose than for the trisaccharide linked to Synsorb, and was several times more effective in the immunolysis of trypomastigotes than the corresponding antiGal from normal human serum. Lytic antibodies were partly absorbed by Serratia marcescens but not by Escherichia coli O111. A human volunteer immunized with an S. marcescens vaccine elicited a specific antiGal response that was lytic to trypomastigotes (70% lysis). We suggest that in vivo high-affinity antiGal antibody clones, as occur in Ch patients, may significantly contribute to the destruction of the parasite, whereas low-affinity antiGal clones are much less effective in the protection against T. cruzi infection.     

Treatment of a murine B cell lymphoma with monoclonal antibodies and IL 2

A transplantable murine B cell lymphoma was used to study combination therapy with anti-idiotype antibody and interleukin 2 (IL 2). Class-switched IgG2a and IgG2b antibodies were compared. A marked additive and sometimes synergistic effect was seen when IL 2 was combined with either IgG2a or IgG2b anti-idiotype antibodies. A synergistic effect was also seen when similar experiments were performed in nude mice. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays showed that IL 2 enhanced antibody-mediated lysis by peritoneal cells exposed to IL 2 in vitro in a dose-related manner. Peritoneal cells harvested from mice treated in vivo with IL 2 contained an increased number of T cells and asialo GM+ natural killer cells, and also mediated enhanced ADCC. Depletion of natural killer cells with anti-asialo GM and complement resulted in a marked decrease in the antibody-dependent cytotoxicity mediated by these peritoneal cells. The mechanism of synergy between monoclonal antibody and IL 2 may be due to the direct or indirect activation of natural killer cells mediating ADCC.     

伤寒Vi多糖结合疫苗免疫小鼠后的抗体类型及动态分析

伤寒Vi多糖结合疫苗和Vi多糖疫苗分别免疫小鼠,分离血清,采用间接ELISA法测定不同时点血清中特异性IgA、IgM、IgG及其亚类(IgG1、IgG2a、IgG3)的抗体滴度。结果显示,免疫一针后,Vi多糖结合疫苗组的IgG抗体GMT值明显升高,第二针有加强效应(P<0.01);所测3种IgG亚型中IgG2a抗体滴度升高明显;Vi多糖和结合疫苗免疫小鼠后,血清中IgA和IgM抗体滴度均有显著升高,但无加强应答。显示Vi多糖结合疫苗在诱导小鼠血清IgG应答方面有加强效应。     

Functional activity of different IgG subclass antibodies against type b capsular polysaccharide of Haemophilus influenzae

The biologic activity of different human IgG subclass antibodies directed against the Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was compared by using an in vitro complement-mediated bactericidal assay and an in vivo passive protection assay in infant rats. An IgG pool was made by Sephacryl S-300 chromatography of sera from adults immunized with PRP vaccine. An IgG2 subclass fraction was prepared by column immunoabsorption of the IgG pool with anti-IgG1 monoclonal antibody. An IgG1 subclass fraction was eluted from the affinity matrix. IgG1, IgG2, IgG3, and IgG4 concentrations in the fractions were measured by solid-phase competitive radioimmunoassays, and anti-PRP antibody was measured by a modified Farr assay. Each fraction was greater than 90% pure IgG2 or IgG1, respectively. There were no significant differences in the minimal anti-PRP antibody concentrations required to kill 50% of Hib cells in vitro (IgG, 0.22; IgG1, 0.21; and IgG2, 0.42 microgram/ml). Similarly, equivalent amounts of anti-PRP antibody of the IgG1 or IgG2 fractions protected against bacteremia (IgG1, 0.12; IgG2, 0.24 microgram per rat). IgG absorbed to remove anti-PRP antibody was neither bactericidal nor protective. Thus IgG1 and IgG2 anti-PRP antibody have equivalent functional activities against Hib as determined by these biologic assays.     

Antigenic relationships between Candida albicans and various yeasts as reflected by immunoglobulin-class specificity.

A group of guinea pigs was inoculated into the foot pads with a single dose of Candida albicans in complete Freund's adjuvant, while another group was similarly inoculated once in the foot pads but also several times intramuscularly, with Candida alone. All guinea pigs were bled at different intervals after immunization and sera were separated chromatographically into IgG and IgM fractions. In order to study the antigenic relationships as reflected by immunoglobulin-class specificity, IgG and IgM fractions and whole sera obtained from guinea pigs differently immunized, were tested for the presence of agglutinins against C. albicans, six other species of Candida, and species of the ascosporogenous genera Saccharomyces, Kluyveromyces and Schizosaccharomyces. The results show that (1) only IgG fractions of the different sera prepared contained the specific anti-C. albicans antibodies; (2) IgG and IgM fractions of the sera obtained from a single inoculation did not reveal a specific pattern expressing antigenic relationships of the yeast studied, and (3) the IgM fractions of the sera obtained from several inoculations had a more homogenous pattern of reactivity, since mainly these contained the agglutinins against the ascosporogenous yeast species.