DEVELOPMENT OF DIAGNOSTIC PAN-ENTEROVIRUS MONOCLONAL ANTIBODIES USING VP1 RECOMBINANT PROTEINS

Lynn Yihong Miao, Christina Pierce, Sarah Peaslee, Carl Shaw, April Brandon, Nate Chapman, Jill DeLotell, Jimmy Page, David SchollDiagnostic HYBRIDS, Athens, OH

AbstractThe VP1 genes of Coxsackie virus B3 (Cox B3), Echovirus 30 (Echo 30) and Poliovirus 1 (Polio 1) were cloned into pQE60 expression vector respectively, and their corresponding recombinant proteins were expressed in E.coli and purified by affinity chromatography. The three recombinant proteins were used to immunize Balb/c mice in an attempt to raise hybridoma monoclonal antibodies (mAb) against a broad range of enteroviruses. After fusion of VP1 proteins primed splenocytes with myeloma cell line, we screened hybridomas against a panel of enterovirues by both ELISA and IFA. Several Polio 1 VP1 derived mAbs and Cox B3 derived mAbs were identified as pan-enterovirus mAbs, which showed ability to recognize most echovirus and coxsackie virus strains, as well as all three poliovirus strains. Based on the IFA screening result, one of each Cox B3 VP1 and Polio1 VP1 derived pan-enterovirusmAbs were selected for further characterization studies on their binding affinity, epitope analysis and cross-reactivity. Both pan-enterovirus mAbs exhibited binding to all three different VP1 proteins in ELISA as well as in Western blotting. The results of competition ELISA and peptide-based ELISA indicates that the two pan-enterovirus mAbs recognize a group common epitope amongst enterovirus VP1 proteins. The mAbs were tested on 120 clinical isolates, and an equivalent or better IFA staining was observed when compared to a predicate device. Except a weak cross reaction to adenovirus 14, the pan-enterovirus mAbs were tested negative against 20 host cell lines, 50 viral strains, and 20 bacteria strains. The pan-enterovirus mAbs has been blended with additional enterovirus 70, enterovirus 71 and Cox A16 specific mAbs as a D3 IFA Enterovirus ID Kit.

BackgroundEnteroviruses, comprising 64 serotypes, cause a wide range of diseases in humans. Diagnosis of enterovirus infection mainly depends upon laboratory testing since the clinical symptoms of the virus infection are difficult to distinguish from other diseases. Isolation of the virus in cell culture followed by IFA protocol has been routinely used for thelaboratory diagnosis of enteroviruses. However, the availability of high quality mAb reagents for IFA is very limited due to the large antigenic diversity amongst enteroviruses. Current commercial pan-enterovirusmAb reagents used in clinical labs have shown either lack of coverage of enteroviruses or cross-reactivity to many other non-enteroviruses. Research study on a common epitope region found in the VP1 proteins of enteroviruses has provided the possibility for us to develop higher quality enterovirus speicific pan-enterovirus mAbs for diagnosis of a broad range of enterovirus infections.

MethodsThe VP1 genes of Cox B3 and Echo 30 were amplified from the viral RNA by RT-PCR. Polio 1 VP1 gene was synthesized by assembly PCR based on its nucleotide sequence in GenBank. After sequence confirmation, the three VP1 genes were subcloned into pQE60 vector respectively. The corresponding recombinant proteins encoded by the three VP1 genes were expressed in E.coli and purified by affinity chromatography. The recombinant VP1 proteins were used to immunize BALB/c mice, and fusions of the VP1 protein primed splenocytes with myeloma cells (SP2/0, Ag14) were carried out. Hybridomas were screened by ELISA and IFA on the target enterovirus infected cell monolayers. The positives were further screened on enterovirus panel slides (Bion) to identify pan-enterovirus mAb producing hybridomas. Several pan-enterovirus mAbs were obtained and characterized by indirect ELISA, competition ELISA, Western blotting, and microscopy for their binding affinity, cross-reactivity and epitope analysis .

Mkr

Polio

1VP1

CoxB3V

P1Ech

o30V

P1

Fig.1 Expression and purification of enterovirus VP1 proteins. (a). Amino acid sequences encoded by three VP1 genes cloned from Coxsackievirus B3, poliovirus 1 and echovirus 30 were aligned. The sequences showing a high degree of homology are highlighted. (b). Affinity purified recombinant proteins expressed from the three enterovirus VP1 genes were analyzed on an SDS-PAGE gel. The proteins were used to immunize mice for raising hybridoma mAbs

(a)

(b)

Fig. 2 Reactivity of Pan-enterovirus MAbs to Four Different Enteric Virus VP1 Proteins.The graphs depict four parameter fits of the data obtained from an indirect binding ELISA. MAbs derived from Coxsackie B3 VP1 and polio 1 VP1 were titrated on immobilized VP1 proteins of four different enterovirusesrespectively. The binding of the mAbs to the immobilized proteins was detected by anti-mouse IgG-HRP conjugate. The results demonstrated that the both mAbs are able to recognize three distinct enterovirus VP1 proteins with different binding affinity, while almost no binding was observed on enterovirus 70 VP1 protein.

Binding of Coxsackie B3 VP1 Derived MAb to Four Distinct Enteric Virus VP1 Proteins

Binding of Poliovirus 1 VP1 Derived MAb to Four Distinct Enteric Virus VP1 Proteins

! Echo 30 VP1 ! Polio 1 VP1! Coxsackie B3 VP1! Entero 70 VP1

! Echo 30 VP1 ! Polio 1 VP1! Coxsackie B3 VP1! Entero 70 VP1

Coxsackie B3 VP1 mAb Polio 1 VP1 mAb

40KD

Fig. 3 Western blot analysis of two enteric virus VP1 mAbs. MRC-5 cells infected with Coxsackievirus B3 or echovirus 30, and Vero cells infected with poliovirus 1 were lysed. The cell lysates were run in SDS-PAGE gels for Western blotting. The mAbs derived from Coxsackie B3 VP1 and polio 1 VP1 were used for the protein detection. The results exhibit that both mAbs are able to recognize the VP1 proteins present in three different enteroviruses.

! Coxsackie B3 VP1 mAb" Polio 1 VP1mAb# Entero 70 VP1 mAb

Fig. 5 Epitope analysis of the Pan-Enterovirus mAbs. A peptide was synthesized based on the VP1 group common epitope sequence “PALTAVETGATNPL”. MAbs derived from Coxsackie B3 VP1 and polio 1 VP1 were incubated with immobilized the peptide in an indirect ELISA. The binding of the mAbs to the immobilized peptide was detected by anti-mouse IgG-HRP conjugate. The graphs depict four parameter fits of the data obtained from the ELISA. The result shows that both mAbs recognize the VP1 group common peptide but with very different binding affinity, suggesting the binding site of the two VP1 mAbs may not exactly the same.

Fig. 4 Competition between Coxsackie B3 VP1 mAb and polio1 VP1 mAb for binding of echo 30 VP1 protein. The graphs depict four parameter fits of the data obtained from an competition ELISA. Biotinylated Coxsackie B3 VP1 mAb was incubated with immobilized echo 30 VP1proteins in the presence of different concentration of unlabeled polio 1 VP1 mAb (unlabeled Coxsackie B3 VP1 mAb and normal mouse IgG were used as control). The binding of the biotinylated Coxsackie B3 VP1 mAbs to the immobilized proteins was detected by neutralite avidin-HRP. The result shows that polio VP1 mAb competes with Coxsackie B3 VP1 mAb for binding of echo 30 VP1 protein, indicating the two mAbs may recognize the same group common epitope of enterovirus VP1 proteins.

# Polio VP1 mAb# Coxsackie B3 VP1 mAb$ Normal mouse IgG

Table/Image 1. Detection of enteroviruses with combined Coxsackie B3 VP1 mAb and polio 1 VP1 mAb. Monolayer of Super E - Mix cells were infected with different strains of enterovirus clinical isolates for 24 hrs. The cell monolayers were stained with the combined pan-enteroivrus mAbs by IFA after fixation with 80% acetone. Poliovirus panel slide from Bion was used to take the images. The images were required at 100x magnification using a Nikon TS 100 microscope with a FITC wide pass filter set. The results demonstrated that the two pan-enterovirus mAbs are able to detect a broad range of entroviuses.

Coxsackie virus Echovirus Poliovirus Enterovirus

A9 A16 B1 B2 B3 B4 B5 B6 4 5 6 7 9 11 13 14 18 20 30 34 1 2 3 69 70 71

Coxsackie B3 VP1 mAb + - + + + + + + - + + + + + + + + + + + + + + + - -

Poliovirus 1 VP1 mAb + - + + + + + + + + + + + + + + + + + + + + + - - -

DHI D3 IFA Enterovirus ID Kit + + + + + + + + + + + + + + + + + + + + + + + + + +

Enterovirus mAb

1. Pan-enterovirus mAbs have been developed using Polio 1 and Coxsackie B3 VP1 recombinant proteins. 2. The mAbs recognize a group common epitope among enterovirus VP1 proteins, and are able to identify Echo

strains, Coxsackie strains and all three Polio strains specifically. 3. The pan-enterovirus mAbs has been blended with additional enterovirus 70, entrovirus 71 and Coxsackie A16

specific antibodies as a D3 IFA Enterovirus ID Kit. 4. The kit has exhibited great ability to identify a broad range of enteroviruses including enterovirus 70, 71 and

Coxsackie A16, without showing cross reactivity to non-enteroviruses such as HSV and CoV.

Summary of Results