CVI Accepts, published online ahead of print on 26 November 2014 Clin. Vaccine Immunol. doi:10.1128/CVI.00519-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Characterization of cross-reactive norovirus-specific monoclonal antibodies

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Baijun Koua, Sue E. Crawforda, Nadim J. Ajamia, Rita Czakóa, Frederick H. Neilla, Tomoyuki N.

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Tanakad, Noritoshi Kitamotoe, Timothy G. Palzkilla,b, Mary K. Estesa,c, Robert L. Atmara,c#

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Departments of Molecular Virology & Microbiologya, Pharmacologyb, and Medicinec, Baylor

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College of Medicine, Houston, Texas, U.S.A.; Sakai City Institute of Public Health, Sakai,

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Japand, Department of Food Science and Nutrition Himeji College of Hyogo, Himeji, Japane

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Running Head: Characterization of Norovirus-specific MAbs

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Address Correspondence to:

Robert L. Atmar, M.D.

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1 Baylor Plaza, MS BCM280

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Houston, TX 77030

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Phone:

(713) 798-3849

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FAX:

(713) 798-6802

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Email:

[email protected]

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Abstract:

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Noroviruses (NoVs) commonly cause acute gastroenteritis outbreaks. Broadly reactive

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diagnostic assays are essential for rapid detection of NoV infections. We previously generated a

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panel of broadly reactive monoclonal antibodies (MAbs). We characterized MAb reactivity with

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NoV virus-like particles (VLPs) from 13 different genotypes (GI=5, GII=8) coated on a

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microtiter plate (direct ELISA) and by western blot. MAbs were genotype-specific or

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recognized multiple genotypes within a genogroup and between genogroups. We next applied

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surface plasmon resonance (SPR) to measure MAb dissociation constants (Kd) as a surrogate for

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binding affinity; a Kd level less than 10 nM was regarded as strong binding. Some MAbs did not

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interact with the VLPs by SPR. To further assess this lack of MAb-VLP interaction, the MAbs

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were evaluated for their ability to identify NoV VLPs in a capture ELISA. Those MAbs for

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which a Kd could not be measured by SPR also failed to capture the NoV VLPs; in contrast,

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those with a measurable Kd gave a positive signal in the capture ELISA. Thus, some broadly

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cross-reactive epitopes in the VP1 protruding domain may be partially masked on intact particles.

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One MAb, NV23, was able to detect genogroup I, II and IV VLPs from 16 genotypes tested by

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sandwich ELISA, and it successfully detected NoVs in stool samples positive by real-time

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reverse transcription-PCR when the Ct value was 31 (GII.17,

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Ct = 35.4). The Ct values for the other positive samples ranged from 19.2 to 29.7 (mean 25.1).

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All virus-negative stools tested (n=12) were also negative in the antigen detection ELISA.

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Discussion:

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MAbs are important reagents in the development of viral diagnostic tests, and a number of NoV

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antigen detection assays utilize MAbs in their kits, including NS14 and 3912 described here (23-

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25). In the current study we further characterized a panel of NoV-specific MAbs, many of which

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recognize common or overlapping linear epitopes in the C-terminal P1 domain of the virus

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capsid (S. E. Crawford, N. J. Ajami, T. D. Parker, N. Kitamoto, K. Natori, N. Takeda, T. Tanaka,

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B. Kou, R. L. Atmar, and M. K. Estes, companion paper submitted for publication). Several of

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the MAbs were unable to recognize VLPs in suspension such as when assayed by SPR or capture

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ELISA. One MAb, NV23, recognized all NoV genotypes tested, and it also was able to detect

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NoVs in clinical fecal samples.

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The direct ELISA format described here is a common means for screening MAbs for reactivity

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to NoV antigens. A number of investigators have identified MAbs that recognize NoV strains

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from many different epitopes. These include epitopes that are in the shell domain (12,13) and

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others that are in the protruding domain (11,26,27) of the capsid protein. Relatively few reports

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characterizing the reactivity of broadly reactive MAbs have described the detection of virus in

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clinical samples (14,28,29), other than in the description of commercially-marketed assays. We

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hypothesize that when NoV VLPs are adsorbed to microtiter plates, conformational changes

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occur that expose epitopes that are masked when the VLPs or virus particles are in suspension.

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Similar findings have been reported in other virus systems in which adsorption on plastic is

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associated with disruption of virus structure and partial denaturation of viral proteins, leading to

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exposure of cryptic epitopes (30,31). The results from this study and our unpublished experience

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in unsuccessfully exposing non-surface exposed NoV epitopes suggest that future reports should

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include information on detection of VLPs in suspension or native virions when characterizing

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broadly reactive MAbs or other detection ligands.

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All of the broadly cross-reactive MAbs in this report map to the C-terminal P1 domain of the

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VP1 capsid protein (11). NV23, NV37 and NV3 recognized all of the VLPs evaluated by direct

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ELISA (Table 4), and these MAbs were shown by Crawford et al. (submitted for publication) to

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compete with each other in competition ELISAs. However, neither NV3 nor NV37 recognize

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VLPs in suspension using a capture ELISA or SPR analysis, suggesting the epitope(s)

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recognized by these MAbs is distinct from that recognized by NV23. Similarly, NS22 competes

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with NV23 but primarily recognizes GII strains. Thus, there appear to be several closely linked

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but distinct epitopes in this region of the capsid.

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The broadly-reactive MAbs that only recognize masked epitopes (e.g., NV3) may also be of

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value. For example, these epitopes may become unmasked during inactivation procedures, such

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as during thermal or chemical treatment. Enhanced detection of epitopes has been reported

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previously following heat or chemical inactivation of HIV-1 (32). Detection of signal with these

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MAbs would indicate a conformational change occurred in the virus capsid protein and might be

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monitored over time to examine inactivation kinetics. These MAbs also would be useful for

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virus capsid detection in immunochromatography studies.

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SPR has been proposed as a method for selecting and characterizing MAbs for diagnostic studies

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(33); however, such studies have not been reported for candidate norovirus-specific MAbs. We

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observed that the measurement of a Kd by SPR was a more sensitive measure for identifying the

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ability of MAb to interact with an intact VLP or virion than was the capture ELISA used in the

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current study (Figure 3), where polyclonal antisera were used for capture. For NV23, Kd values

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for MAbs that interact with all the tested NoV genotypes were

Characterization of cross-reactive norovirus-specific monoclonal antibodies.

Noroviruses (NoVs) commonly cause acute gastroenteritis outbreaks. Broadly reactive diagnostic assays are essential for rapid detection of NoV infecti...
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