Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus: bacterial expression and refolding.

Single chain antibodies (ScFvs) are heavy and light chain variable domains connected by an artificial linker. Because of their smaller size, ScFvs show improved tissue penetration in vivo and reduced immunogenicity, making them ideal for therapeutic applications. We have cloned a ScFv against western equine encephalitis (WEE) using rDNA technology. The ScFv was generated from a hybridoma cell line (11D2) specific to the WEE virus E1 glycoprotein and is arranged in the V(L)-V(H) orientation with a (gly(4)ser)(3) linker. This ScFv was engineered successfully with a biotin mimic tag (11 amino acid peptide) and cloned in the pET22b+ expression vector. The ScFv was expressed as a approximately 32kDa protein in Escherichia coli as inclusion bodies, with an estimated yield of 20-40 mg/l. Different refolding protocols were used to solubilise the inclusion bodies. Most of the functional ScFv was generated when the inclusion bodies were solubilized in a detergent, air oxidised in the presence of CuSO(4) and then denatured in urea buffer in comparison to other protocols. The product was renatured finally in Tris arginine buffer (pH 8.0). Refolded protein was dialysed against phosphate buffer saline (PBS) (pH 7.3) to remove the Tris and arginine. Our refolding protocol generated up to a 50% yield of soluble protein, which retained antigen-binding activity with whole inactivated WEE virus as demonstrated by ELISA and Western blot analysis. This 11D2-biotin mimic ScFv complexed with streptavidin horseradish peroxidase (St-HRPO) will be useful as a detector reagent in the ultrasensitive ELISA detection of WEE virus antigen.

Development of a second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: expression and functional analysis.

We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.

Bispecific and bifunctional single chain recombinant antibodies.

Bispecific and bifunctional monoclonal antibodies as second generation monoclonals, produced by conventional chemical or somatic methods, have proved useful in the immunodiagnosis and immunotherapy of cancer and other diseases. Recombinant antibodies produced by genetic engineering techniques have also become available for use in preclinical and clinical studies. Furthermore, through genetic engineering, it is possible to remove or add on key protein domains in order to create designer antibody molecules with two or more desired functions. This review summarizes the strategies for development of single chain variable fragment (scFv) bifunctional and bispecific antibodies. The advantages and disadvantages as well as the problems of generating the various bispecific and bifunctional antibody constructs are reported and discussed. Since conventionally prepared bispecific and bifunctional monoclonal antibodies have already shown promise in clinical trials and results from preclinical studies of recombinant bispecific antibodies are encouraging, clinical trials in humans of recombinant bispecific and bifunctional antibodies, as a new generation of biologicals, are likely to be the thrust in the next decade and beyond.

Pharmacokinetics study of a novel chimeric single-chain variable fragment antibody against western equine encephalitis virus.

A novel recombinant single-chain fragment variable (scFv) antibody against western equine encephalitis (WEE) virus has been previously constructed and partially characterized. The RS10B5huFc antibody was made by fusing an anti-WEE scFv to a human heavy-chain IgG1 constant region. The RS10B5huFc antibody was functional in binding to WEE virus in enzyme-linked immunosorbent assays (ELISAs), and the Fc domain of the antibody was capable of effector functions, such as binding to protein G and human complement. In this study, the RS10B5huFc antibody was further characterized by BIAcore analyses and was found to possess a binding affinity to a WEE virus epitope (K[D] = 9.14 x 10(-6) M), 4.5-fold lower than its parental mouse monoclonal antibody (MAb) 10B5 E7E2 (K[D] = 2 x 10(-6) M). No cross-reactivity was found between the RS10B5huFc antibody and three other alphaviruses (Sindbis virus [SIN], Venezuelan equine encephalitis [VEE] virus, and eastern equine encephalitis [EEE] virus). Pharmacokinetics studies showed that the RS10B5huFc antibody (free and encapsulated) was found to be retained in the lungs of mice for greater than 48 h when administered intranasally. In contrast, when administered intramuscularly to mice, the RS10B5huFc antibody was not detected in the lungs and only found in the liver and kidneys.

DNA vaccination against respiratory influenza virus infection.

DNA vaccination using plasmid encoding the hemagglutinin (HA) gene of influenza A/PR/8/34 virus to induce long-lasting protective immunity against respiratory infection was evaluated in this study. Using liposomes as carriers, the efficacy of DNA vaccines was determined using a lethal influenza infection model in mice. Mice immunized intranasally or intramuscularly with liposome-encapsulated pCI plasmid encoding HA (pCI-HA10) were completely protected against an intranasal 5 LD(50) influenza virus challenge. Mice immunized with liposome-encapsulated pCI-HA10, but not naked pCI-HA10, by intranasal administration were found to produce high titers of serum IgA. These results suggest DNA vaccines encapsulated in liposomes are efficacious in inducing complete protective immunity against respiratory influenza virus infection.

Construction and characterization of monoclonal antibodies against western equine encephalitis virus.

A repertoire of mouse monoclonal antibodies (MAbs) against western equine encephalitis virus (WEE) was constructed and characterized. Anti-WEE antibodies were expressed from hybridomas and purified by protein G chromatography. Each of the antibodies was functionally assessed by indirect enzyme-linked immunosorbent assays (ELISAs), Western blotting, and immunoprecipitations. All antibodies bound to WEE antigen in ELISAs, whereas only a subgroup of antibodies was found to be active in Western blotting and immunoprecipitations. A subset of antibodies was found to cross-react with other alphaviruses, such as Sindbis virus (SIN), Venezuelan equine encephalitis (VEE), and eastern equine encephalitis (EEE). Because many of the antibodies were highly reactive to WEE antigen in one or more of the assays, these antibodies are excellent candidates for immunodetection and immunotherapy studies.

Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus.

A novel recombinant single-chain fragment variable (scFv) antibody against Western equine encephalitis virus (WEE) was constructed and characterized. Using antibody phage display technology, a scFv was generated from the WEE specific hybridoma, 10B5 E7E2. The scFv was fused to a human heavy chain IgG1 constant region (CH1-CH3) and contained an intact 6 His tag and enterokinase recognition site (RS10B5huFc). The RS10B5huFc antibody was expressed in E. coli and purified by affinity chromatography as a 70-kDa protein. The RS10B5huFc antibody was functional in binding to WEE antigen in indirect enzyme-linked immunosorbent assays (ELISAs). Furthermore, the RS10B5huFc antibody was purified in proper conformation and formed multimers. The addition of the human heavy chain to the scFv replaced effector functions of the mouse antibody. The Fc domain was capable of binding to protein G and human complement. The above properties of the RS10B5huFc antibody make it an excellent candidate for immunodetection and immunotherapy studies.

Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes.

The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucleotide differences in 8624 bases (1.7% divergence), of which only 28% (42 nucleotides) altered the encoded amino acids. Comparison of deduced nsP1 and nsP4 amino acid sequences from WEE with the corresponding proteins from eastern equine encephalitis virus (EEE) yielded identities of 84.9 and 83.8%, respectively. Previously uncharacterized stem-loop structures were identified in the nontranslated terminal regions. A cDNA clone of the 26S region encoding the structural polyprotein of WEE strain 71V-1658 was placed under the control of a cytomegalovirus promoter and transfected into tissue culture cells. The viral envelope proteins were functionally expressed in tissue culture, as determined by histochemical staining with monoclonal antibodies that recognize WEE antigens, thus, forming the initial step in the investigation of subunit vaccines to WEE.

Development of a functional monoclonal single-chain variable fragment antibody against Venezuelan equine encephalitis virus.

We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.

A single chain Fv specific against Western equine encephalitis virus.

A recombinant single chain Fv (scFv) specific against Western equine encephalitis virus (WEE) was developed and characterized. The scFv was generated from 11D2 hybridoma producing anti-WEE antibody reactive to E1 component of viral envelope glycoprotein. V(L) and V(H) gene segments of 11D2 scFv were generated and joined together with a (gly4ser)3 linker by polymerase chain reaction (PCR). The resulting scFv was successfully expressed in P. pastoris expression system. Fifteen individual plasmids were tested and six of them were shown to drive scFv expression. DNA sequence analysis from three productive plasmids showed that they all carried the same VL and V(H) gene segments with a few base differences. Comparison of 11D2 scFv DNA sequence to the Kabat database showed that VH of 11D2 antibody belonged to subgroup IIID and subfamily XIV, while VL domain did not belong to any known subgroup or subfamily. Western blot analysis of 11D2 scFv using anti-c-myc antibody for detection showed different band pattern among clones derived from different plasmids. This was thought to be due to the different glycosylation where amino acid substitution occurred. Successful purification of 11D2 scFv could be done by immobilized metal affinity chromatography with an unoptimized yield of 700 microg/L. Functional studies showed that 11D2 scFv could bind to its respective WEE antigen as demonstrated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The binding affinity of 11D2 scFv is reasonably good compared to the parental 11D2 bivalent monoclonal antibody (MAb). Thus, 11D2 scFv and its derivatives have a potential use as immunotherapeutic and immunodiagnostic agents of WEE infections.

Development of a murine hypothermia model for study of respiratory tract influenza virus infection.

A hypothermia model was developed to predict mortality and morbidity caused by respiratory influenza virus infection in mice. To increase virulence, egg-propagated influenza A/PR/8 virus was adapted for growth in mice by four blind serial passages. The mouse-adapted influenza A virus was then used to infect groups of BALB/c mice via the intranasal route, and the 50% lethal dose (LD50) was determined. Rectal temperature of the infected mice was monitored daily, and survival rate was determined at day 14 after infection. The lowest average body temperature recorded in infected mice was approximately 10 degrees C below that in noninfected mice. In mice that developed hypothermia, with body temperature of 32 degrees C or lower, morbidity and mortality inevitably occurred. In this study, the LD50 and the 50% hypothermia-inducing dose (HID50) for mouse-adapted influenza A virus were compared and calculated to be at the same dose. These results suggest that the HID50 model could be used to predict mortality and morbidity associated with influenza virus infection in mice. This model could potentially be used to substantially reduce the time and extent of suffering inflicted on experimental animals due to viral infections, and therefore may serve as a more humane alternative to LD50 determinations.