Isolation and optimization of camelid single-domain antibodies: Dirk Saerens' work on nanobodies

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from all other species, these special antibodies are devoid of light chains, and are composed of a heavy chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol. These HCAbs are easily purified from serum, and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. The antigen binding site of the dromedary HCAb comprises one single domain, referred to as VHH or nanobody (Nb), therefore, a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens. The monoclonal Nb is produced well in bacteria, is very stable and highly soluble, and it binds the antigen with high affinity and specificity. Currently, the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors, to diagnose infections, or to treat diseases such as cancer or trypanosomiasis.     

Quantitative determination of titers of anti-zona serum.

The titers of rabbit antiserum against isolated mouse zonae pellucidae were examined by several methods in connection with inhibitory effect on fertilization. The titers determined by zona precipitate, zona dissolution, indirect immunofluorescence and in vitro fertilization tests were 2(4), 2(1) to 2(4), 2(7) and 2(4), respectively. Cytotoxic effect could not be detected from zona antibody. The indirect immunofluorescence was most sensitive for detection of zona antibody but did not represent the extent of inhibitory effect on fertilization. The titer obtained by zona precipitate test was most close to the titer obtained by inhibitory effect on in vitro fertilization. The present study also demonstrated that at least 0.0375 ml of antiserum per female mouse, equivalent to 0.15-0.25% of body weight, was necessary for inhibition of fertilization in vivo by passive immunization with anti-zona serum.     

Immunization with Treponema pallidum outer membrane vesicles induces high-titer complement-dependent treponemicidal activity and aggregation of T. pallidum rare outer membrane proteins (TROMPs).

The purpose of this study was to determine whether immunization with purified outer membrane vesicles (OMV) from Treponema pallidum (T.p. ) could elicit Abs capable of killing this organism. It is well established that the immunization of rabbits or mice with killed T.p. or with recombinant T.p. Ags has failed to generate serum killing activity comparable with that of infection-derived immunity. Because of the small amount of T.p. OMV obtainable, a single mouse was immunized with purified OMV. The mouse anti-OMV serum and infection-derived immune rabbit serum (IRS) were compared by reactivities on two-dimensional T.p. immunoblots and by the T.p. immobilization test, a complement-dependent killing assay. Whereas IRS detected >40 Ags, the anti-OMV serum identified only 6 Ags corresponding to proteins identified previously in the outer membrane. T.p. immobilization testing showed that IRS had a 100% killing titer of 1:44 and a 50% killing titer of 1:662. By comparison, the mouse anti-OMV serum had a significantly greater 100% killing titer of 1:1,408 and a 50% killing titer of 1:16,896. Absorption of the anti-OMV serum to remove Ab against outer membrane-associated lipoproteins did not change the 100% killing titer. Freeze-fracture analysis of T.p. incubated in IRS or anti-OMV serum showed that T.p. rare membrane-spanning outer membrane proteins were aggregated. This is the first demonstration of high-titer killing Abs resulting from immunization with defined T.p. molecules; our study indicates that the targets for these Abs are T. p. rare outer membrane proteins.     

Oral vaccination against Helicobacter pylori with recombinant cholera toxin B-subunit

BACKGROUND: The innocuous pure recombinant cholera toxin B-subunit (rCTB) is very attractive as a strong adjuvant for host immunization, but little is known about rCTB's gastric mucosal immunoadjuvanticity against Helicobacter pylori. The immunoadjuvanticity of rCTB against H. pylori was tested. MATERIAL AND METHODS: Mice were immunized with sonicated H. pylori and rCTB orally or intranasally and sacrificed on day 42 after immunization. Passive cutaneous anaphylaxis (PCA) test was performed to evaluate IgE-mediated anaphylaxis with serum from mice to which H. pylori-antigen with rCTB had been administered. Immunoglobulin titer specific to H. pylori in serum, lavation of the gastrointestinal tracts and feces were examined. Gastritis in vaccinated mice after a challenge was assessed with the scoring defined from grading of gastric inflammation. H. pylori proliferation after immunization was investigated by counting colony forming units (CFU) per gram of stomach tissue. RESULTS: PCA test exhibited no reactions against the serum from mice immunized with H. pylori-antigen with rCTB administered orally and intranasally. Oral and nasal coadministrations of rCTB significantly raised systemic and mucosal immunities against H. pylori and suppressed proliferation of H. pylori in gastric mucosa. The score of gastritis in mice immunized orally was significantly higher than that of mice immunized nasally due to postimmunization gastritis. Only oral administration of rCTB suppressed H. pylori proliferation as compared with intranasal administration and without rCTB. CONCLUSIONS: The present study indicated that rCTB has systemic and mucosal immunoadjuvanticities against H. pylori and that oral vaccination with rCTB might additively support antibiotic eradication.     

Prospects for the control of sheep blowfly strike by vaccination

Research into vaccination against flystrike is aimed at either controlling the predisposing condition, fleece rot, or direct control of the fly maggots. A vaccine against the major bacterial species found in fleece rot lesions, Pseudomonas aeruginosa, is undergoing field trials and results suggest that this vaccine may reduce fleece rot incidence. Problems to be investigated include the existence of variants of P. aeruginosa in the field and the involvement of other species of bacteria in fleece rot. Strategies for direct vaccination include immunization with larval products involved in wound formation and larval nutrition and immunization against novel antigens usually from the gut of first instar larvae. Both methods have resulted in significant inhibition of larval growth. Analysis of larval products has revealed a number of active proteases which degrade skin proteins such as collagen. Inhibition of these enzymes with plasma enzyme inhibitors also affects larval growth in vitro. Antibodies raised against these enzymes are being tested for inhibitory effects against larvae and used to isolate cDNA clones from Lucilia cuprina libraries. Antigens from the gut are able to induce antibodies inhibitory to larval growth both in vitro and in vivo. Isolation of these antigens is proceeding in a number of laboratories. Problems still to be analysed include whether growth inhibition produces effective protection in the field and whether sufficient antibody will have early access to the larvae to significantly affect them.     

Can the surface immobilization antigens of Philasterides dicentrarchi (Ciliophora: Scuticociliatida) be used as target antigens to develop vaccines in cultured fish?

In order to test whether immobilization antigens (i-antigens) of Philasterides dicentrarchi could be suitable antigenic targets against scuticociliatosis, polyclonal olive flounder (Paralichthys olivaceus) sera were raised against P. dicentrarchi by immunization with lysates of ciliates grown using chinook salmon epithelial (CHSE) cells, and the ability of the immune sera to kill the ciliates via classical complement pathway was analyzed in relation to agglutination activity. The immune sera showed clear agglutination activity against the CHSE-cultured ciliates. However, the agglutinated ciliates were not killed but escaped from the agglutinated mass within a few hours. Ciliates isolated from fish artificially infected with the same population of CHSE-cultured ciliates were not agglutinated by the immune sera even at the lowest dilution. In antibody-dependent complement-mediated killing (ADCK), the immune sera completely killed the CHSE-cultured ciliates at relatively higher serum dilutions (showing low or no agglutination activity). However, CHSE-cultured ciliates were not killed completely at lower immune serum dilutions (showing high agglutination activity). In contrast to CHSE-cultured ciliates, the ciliates isolated from infected fish were killed at lower dilutions of the immune sera in spite of no agglutination response. Considering the presence of various i-antigen types, ability to change i-antigen type in response to corresponding antibody, and relatively low ADCK activity at high agglutination titer, i-antigens of P. dicentrarchi may not be good targets for subunit vaccine development. To develop subunit vaccines against scuticociliatosis, other surface antigens expressed constitutively or expressed specifically under the infection state for survival of the ciliates in the host fish might be more favorable to elicit protective antibodies than the surface i-antigens.     

Lack of development of phagocytic inhibitory activity in serum of cats immunized withEnterobacter cloacae lipopolysaccharide

Sequentially collected sera from cats immunized withEnterobacter cloacae lipopolysaccharide (LPS) were assessed for their effect on phagocytosis by incubating alveolar marcophage monolayers in the presence of³H-labeled bacteria and 5% serum from control or immunized animals. Unlike serum fromPseudomonas aeruginosa LPS-immunized orP. aeruginosa-infected cats in previous studies, which contained phagocytic inhibitory activity specific forP. aeruginosa, serum from 12 of 13 cats immunized withE. cloacae LPS did not contain phagocytic inhibitory activity forE. cloacae orP. aeruginosa. An enzyme-linked immunosorbent assay (elisa) demonstrated a significant rise inE. cloacae LPS specific IgG (peak titer 1:10,240) by week 13 after immunization. This study suggests that long-term immunization withE. cloacae LPS does not result in the induction of macrophage phagocytic inhibitory activity as previously demonstrated withP. aeruginosa LPS immunization.     

Heavy-chain antibodies in<Emphasis Type="Italic"> Camelidae</Emphasis>; a case of evolutionary innovation

The emergence in Camelidae species of functional antibodies devoid of light chains (referred to as heavy-chain antibodies or HCAbs) is an intriguing evolutionary event. Homodimeric HCAbs have also been documented in spotted ratfish ( Cos5-Abs) and nurse shark (NAR). To reveal the evolutionary history of HCAbs, we evaluated the phylogenetic and phenotypic relationships among HCAbs and conventional antibodies across taxa and confirmed the current viewpoint that different groups of HCAbs have evolved independently in the three lineages. At least, in the camelids, HCAbs are not the result of resuscitation of dormant genes. They are derived from the conventional antibodies within the Camelidae lineage, and are apparently the outcome of more recent adaptive changes occurring in the compartment of heteromeric antibodies. The shared structural properties of HCAbs across taxa are therefore explained by convergent evolution due to similar constraints related to the absence of pairing to the light chain. It appears that innovative evolutionary changes in Camelidae have led to a new level of antigen binding repertoire diversification and have allowed acquisition of novel antigen-receptor properties.     

Single domain antibodies: a new concept for epidermal growth factor receptor and EGFRvIII targeting

Epidermal growth factor receptor (EGFR) is one of the major molecular targets for cancer diagnosis and therapy. EGFR and EGFRvIII, mutated form of EGFR, have been identified as participating in pathogenesis of some forms of human cancers. Monoclonal antibodies (mAbs) targeting EGFR/EGFRvIII have been shown to suppress the signal transduction pathways controlling tumor cell growth, proliferation, and apoptosis. Until now, different types of mAbs or antibody fragments against EGFR family have been established. Some of these antibodies have been used clinically for treating various forms of human malignancies. More recently, a single domain antibody (sdAb) targeting this family of receptors has been introduced. The heavy chain antibodies (HCAbs) that made up variable regions of heavy chain, CH2, and CH3 domains are shown in camelids. SdAbs derived from camel HCAbs are the smallest known natural building parts for binding to antigen. They also possess a longer antigen recognizing region, which increases their capability for being more specific in target antigen enhancement. Camelid antibodies are highly valuable for their special characteristics, including heat resistance, small size, high solubility in an aqueous environment, and non-immunogenicity in a human environment. Due to these abilities, research on biotechnological production and treatment applications of recombinant smaller fragments of these only HCAbs is widely in progress. In this article, we will discuss the challenges and successes of different types of mAbs targeting EGFR/EGFRvIII in human cancer.     

Immunoglobulin G, Plasma Cells, and Lymphocytes in the Murine Vagina after Vaginal or Parenteral Immunization with Attenuated Herpes Simplex Virus Type 2

This investigation evaluated immunity to vaginal herpes simplex virus type 2 (HSV-2) infection after local or parenteral immunization with attenuated HSV-2. Vaginal immunization induced sterilizing immunity against challenge with a high dose of wild-type virus, whereas parenteral immunizations protected against neurologic disease but did not entirely prevent infection of the vagina. Vaginal immunization caused 86- and 31-fold increases in the numbers of immunoglobulin G (IgG) plasma cells in the vagina at 6 weeks and 10 months after immunization, whereas parenteral immunizations did not increase plasma cell numbers in the vagina. Vaginal secretion/serum titer ratios and specific antibody activities in vaginal secretions and serum indicated that IgG viral antibody was produced in the vagina and released into vaginal secretions at 6 weeks and 10 months after vaginal immunization but not after parenteral immunizations. In contrast to the case for plasma cells, the numbers of T and B lymphocytes in the vagina were similar in vaginally and parenterally immunized mice. Also, lymphocyte numbers in the vagina were markedly but similarly increased by vaginal challenge with HSV-2 in both vaginally and parenterally immunized mice. Lymphocyte recruitment to the vagina after virus challenge appeared to involve memory lymphocytes, because it was not observed in nonimmunized mice. Thus, local vaginal immunization with attenuated HSV-2 increased the number of IgG plasma cells in the vagina and increased vaginal secretion/serum titer ratios to 3.0- to 4.7-fold higher than in parenterally immunized groups but caused little if any selective homing of T and B lymphocytes to the vagina.     

Inhibition of protein tyrosine phosphatases by low-molecular-weight S-nitrosothiols and S-nitrosylated human serum albumin

The homogeneous recombinant mammalian protein tyrosine phosphatase 1B (PTP1B) and Yersinia protein tyrosine phosphatase (PTPase) are inactivated by a series of low-molecular-weight S-nitrosothiols. These compounds exhibited different inhibitory activities in a time- and concentration-dependent manner with second-order rate constants (k(inact)/K(I)) ranging from 37 to 113 M(-1) min(-1) against mammalian PTP1B and from 66 to 613 M(-1) min(-1) against Yersinia PTPase. Furthermore, the inactivation of Yersinia PTPase by S-nitrosylated protein:S-nitroso human serum albumin was investigated. Both single-S-nitrosylated and poly-S-nitrosylated human serum albumin show good inhibitory ability to Yersinia PTPase. The second-order rate constants are 472 and 1188 M(-1) min(-1), respectively. This result indicates a possibility that S-nitrosylated albumin in vivo may function as an inhibitor for a variety of cysteine-dependent enzymes.     

重组质粒pcDNA3-dnaJ/蛋白DnaJ异源免疫诱导Th1和Th17细胞免疫应答抵抗肺炎链球菌感染 *

目的 探索更有效的肺炎链球菌DNA疫苗和疫苗免疫策略,并探究其中的保护机制。方法 构建重组质粒pcDNA3-dnaJ并表达DnaJ蛋白,实验分别设置重组质粒pcDNA3-dnaJ/蛋白DnaJ免疫小鼠组及单独质粒pcDNA3-dnaJ免疫小鼠组,分别比较肺炎链球菌菌株攻毒后小鼠鼻腔灌洗液细菌载量及生存率,采用ELISA检测免疫小鼠血清抗体效价及炎症因子,流式细胞术分析体外BMDCs激活情况及Th1和Th17细胞免疫应答。结果 质粒pcDNA3-dnaJ免疫3次可诱导血清中抗原特异性抗体的产生,并减少肺炎链球菌攻毒后鼻咽部的细菌载量,但在防止致死性感染方面效果较差。然而,与重复质粒DNA接种三次相比,pcDNA3-dnaJ 1次/ DnaJ蛋白加强1次的免疫策略可以显著减少鼻咽中的肺炎链球菌定植,并能够更好的预防致死性感染。此外,与DNA质粒加强免疫相比,DnaJ蛋白加强免疫后可产生更高水平的IFN-γ和IL-17A。结论 重组质粒pcDNA3-dnaJ/蛋白DnaJ异源免疫可能通过活化树突状细胞,进而诱导Th1和Th17细胞免疫应答,抵抗肺炎链球菌感染。     

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.     

鸡血清与卵黄中抗中华眼镜蛇毒IgY动态变化研究

目的探索特异性IgY的产生和变化规律。方法用眼镜蛇毒原毒免疫产蛋母鸡,ELISA定期检测卵黄中的抗体效价变化,小鼠体外中和实验检测其生物活性。第1次免疫40周后,眼镜蛇毒攻击已免疫母鸡,检测攻击前后鸡血清中抗体效价变化情况,未经眼镜蛇毒免疫的母鸡作阴性对照。结果经免疫后第7天蛋黄中即可检测到抗体,经多次加强免疫,40周时蛋黄中还能保持高效价的抗体,通过分离纯化,此抗体可保护实验小鼠免受4 LD50眼镜蛇毒的攻击;同时,鸡血清中也保留着较高效价的抗体,可中和4 LD50以上的眼镜蛇毒。结论用眼镜蛇毒免疫鸡,经多次加强免疫,卵黄和鸡血清中可持久保持高效价的特异性抗体,初步检测此抗体可中和4 LD50的蛇毒。     

Live non-invasive Shigella dysenteriae 1 strain induces homologous protective immunity in a guinea pig colitis model

A non-invasive live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed from a Shiga toxin gene deleted mutant of Shigella dysenteriae 1 by introducing a plasmid vector pPR1347 that carried a lipopolysaccharide biosynthesis gene (rfb and rfc) of Salmonella typhimurium. In guinea pigs, four successive oral administrations of LTSH Δstx showed complete protection against rectal challenge with wild type S. dysenteriae 1 strain. Exponential increase of the serum IgG and IgA titer against lipopolysaccharide of LTSH Δstx was observed during immunization, peaked on day 28 and remained at that level until day 35 after the initiation of the immunization. In intestinal lavage of the immunized animals, significant increase of IgA titer against lipopolysaccharide of LTSH Δstx was also observed. These data suggested that LTSH Δstx could be a useful candidate to induce protective immunity against S. dysenteriae 1 infection.     

Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.     

Evidence for shared antigenic determinants on rabbit and human interleukin 1 (IL 1)

An antiserum to human interleukin 1 (IL 1) was prepared by immunizing a goat with the isoelectric point (pI) 6.9 type of IL 1 in Freund's complete adjuvant. Serum-mediated inhibition of the biological activity of IL 1 appeared within 4 wk after the first immunization, and showed a progressive rise in titer over a 9-mo period. The inhibitory moiety was purified by sequential ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography, and the activity was found to co-purify with the IgG fraction of the serum. The antibody neutralized the biological activity of the pI 6.9 type of human IL 1 derived from either human placental tissue or human peripheral blood adherent cells, but did not neutralize the pI 5.2 type of IL 1 derived from either source. When used as an affinity reagent, the antibody selectively absorbed the pI 6.9 human IL 1, but not the pI 5.2 human IL 1. Furthermore, the antibody neutralized the pI 7.4 type of IL 1 derived from rabbit alveolar macrophages, but had no activity against the pI 4.6 IL 1 derived from the same source. No inhibitory activity against rat spleen cell-derived IL 1 or murine P388D1 cell line-derived IL 1 was detected. These experiments support the concept that the differing pI types of IL 1 derived from the same species are both biochemically and antigenically distinct molecules, and IL 1 of similar pI type derived from different species may share antigenic determinants.     

Characteristics of physiological reactions during long-term immunization of rats with protein conjugates of angiotensin II

The results show that during long-term immunization of rats against conjugates of angiotensin II with bovine serum albumin definite age dynamics of immune response have been observed. It was shown that the changes of physiological readings were correlated with growing of antibodies titer against angiotensin II, accordingly, as the immune response was lower, these readings return to the initial level.     

Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential

Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.     

Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract

The roles of IgG and secretory IgA in the protection of the respiratory tract (RT) against influenza infection remain unclear. Passive immunization with Ab doses resulting in serum IgG anti-influenza virus Ab titers far in excess of those observed in immune mice has compounded the problem. We compared the effects of i.v. anti-influenza virus IgG and i.v. anti-influenza virus polymeric IgA (pIgA) mAb administered in amounts designed to replicate murine convalescent serum or nasal Ab titers, respectively. A serum anti-influenza virus IgG titer 2.5 times the normal convalescent serum anti-influenza virus IgG titer was required for detectible Ab transudation into nasal secretions, and a serum IgG titer 7 times normal was needed to lower nasal viral shedding by 98%. Anti-influenza virus pIgA at a nasal Ab titer comparable to that seen in convalescent mice eliminated nasal viral shedding. The RT of influenza-infected pIgA- or IgG-protected mice were studied by scanning electron microscopy. Only pIgA was found to prevent virally induced pathology in the upper RT, suggesting that IgG did not prevent viral infection of the nose, but neutralized newly replicated virus after infection had been initiated. In contrast, IgG, but not pIgA, was found to prevent viral pathology in the murine lung. Our results help to resolve the controversy of IgA- vs IgG-mediated protection of the RT; both Abs are important, with plasma IgG Ab serving as the back-up for secretory IgA-mediated protection in the nasal compartment, and IgG being the dominant Ab in protection of the lung.