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.
Characterization of cross-reactive norovirus-specific monoclonal antibodies
Baijun Koua, Sue E. Crawforda, Nadim J. Ajamia, Rita Czakóa, Frederick H. Neilla, Tomoyuki N.
Tanakad, Noritoshi Kitamotoe, Timothy G. Palzkilla,b, Mary K. Estesa,c, Robert L. Atmara,c#
Departments of Molecular Virology & Microbiologya, Pharmacologyb, and Medicinec, Baylor
College of Medicine, Houston, Texas, U.S.A.; Sakai City Institute of Public Health, Sakai,
Japand, Department of Food Science and Nutrition Himeji College of Hyogo, Himeji, Japane
Running Head: Characterization of Norovirus-specific MAbs
Address Correspondence to:
Robert L. Atmar, M.D.
1 Baylor Plaza, MS BCM280
Houston, TX 77030
Email: [email protected]
Noroviruses (NoVs) commonly cause acute gastroenteritis outbreaks. Broadly reactive
diagnostic assays are essential for rapid detection of NoV infections. We previously generated a
panel of broadly reactive monoclonal antibodies (MAbs). We characterized MAb reactivity with
NoV virus-like particles (VLPs) from 13 different genotypes (GI=5, GII=8) coated on a
microtiter plate (direct ELISA) and by western blot. MAbs were genotype-specific or
recognized multiple genotypes within a genogroup and between genogroups. We next applied
surface plasmon resonance (SPR) to measure MAb dissociation constants (Kd) as a surrogate for
binding affinity; a Kd level less than 10 nM was regarded as strong binding. Some MAbs did not
interact with the VLPs by SPR. To further assess this lack of MAb-VLP interaction, the MAbs
were evaluated for their ability to identify NoV VLPs in a capture ELISA. Those MAbs for
which a Kd could not be measured by SPR also failed to capture the NoV VLPs; in contrast,
those with a measurable Kd gave a positive signal in the capture ELISA. Thus, some broadly
cross-reactive epitopes in the VP1 protruding domain may be partially masked on intact particles.
One MAb, NV23, was able to detect genogroup I, II and IV VLPs from 16 genotypes tested by
sandwich ELISA, and it successfully detected NoVs in stool samples positive by real-time
reverse transcription-PCR when the Ct value was 31 (GII.17,
Ct = 35.4). The Ct values for the other positive samples ranged from 19.2 to 29.7 (mean 25.1).
All virus-negative stools tested (n=12) were also negative in the antigen detection ELISA.
MAbs are important reagents in the development of viral diagnostic tests, and a number of NoV
antigen detection assays utilize MAbs in their kits, including NS14 and 3912 described here (23-
25). In the current study we further characterized a panel of NoV-specific MAbs, many of which
recognize common or overlapping linear epitopes in the C-terminal P1 domain of the virus
capsid (S. E. Crawford, N. J. Ajami, T. D. Parker, N. Kitamoto, K. Natori, N. Takeda, T. Tanaka,
B. Kou, R. L. Atmar, and M. K. Estes, companion paper submitted for publication). Several of
the MAbs were unable to recognize VLPs in suspension such as when assayed by SPR or capture
ELISA. One MAb, NV23, recognized all NoV genotypes tested, and it also was able to detect
NoVs in clinical fecal samples.
The direct ELISA format described here is a common means for screening MAbs for reactivity
to NoV antigens. A number of investigators have identified MAbs that recognize NoV strains
from many different epitopes. These include epitopes that are in the shell domain (12,13) and
others that are in the protruding domain (11,26,27) of the capsid protein. Relatively few reports
characterizing the reactivity of broadly reactive MAbs have described the detection of virus in
clinical samples (14,28,29), other than in the description of commercially-marketed assays. We
hypothesize that when NoV VLPs are adsorbed to microtiter plates, conformational changes
occur that expose epitopes that are masked when the VLPs or virus particles are in suspension.
Similar findings have been reported in other virus systems in which adsorption on plastic is
associated with disruption of virus structure and partial denaturation of viral proteins, leading to
exposure of cryptic epitopes (30,31). The results from this study and our unpublished experience
in unsuccessfully exposing non-surface exposed NoV epitopes suggest that future reports should
include information on detection of VLPs in suspension or native virions when characterizing
broadly reactive MAbs or other detection ligands.
All of the broadly cross-reactive MAbs in this report map to the C-terminal P1 domain of the
VP1 capsid protein (11). NV23, NV37 and NV3 recognized all of the VLPs evaluated by direct
ELISA (Table 4), and these MAbs were shown by Crawford et al. (submitted for publication) to
compete with each other in competition ELISAs. However, neither NV3 nor NV37 recognize
VLPs in suspension using a capture ELISA or SPR analysis, suggesting the epitope(s)
recognized by these MAbs is distinct from that recognized by NV23. Similarly, NS22 competes
with NV23 but primarily recognizes GII strains. Thus, there appear to be several closely linked
but distinct epitopes in this region of the capsid.
The broadly-reactive MAbs that only recognize masked epitopes (e.g., NV3) may also be of
value. For example, these epitopes may become unmasked during inactivation procedures, such
as during thermal or chemical treatment. Enhanced detection of epitopes has been reported
previously following heat or chemical inactivation of HIV-1 (32). Detection of signal with these
MAbs would indicate a conformational change occurred in the virus capsid protein and might be
monitored over time to examine inactivation kinetics. These MAbs also would be useful for
virus capsid detection in immunochromatography studies.
SPR has been proposed as a method for selecting and characterizing MAbs for diagnostic studies
(33); however, such studies have not been reported for candidate norovirus-specific MAbs. We
observed that the measurement of a Kd by SPR was a more sensitive measure for identifying the
ability of MAb to interact with an intact VLP or virion than was the capture ELISA used in the
current study (Figure 3), where polyclonal antisera were used for capture. For NV23, Kd values
for MAbs that interact with all the tested NoV genotypes were