Vaccine 24 (2006) 7056–7065

Animal models and antibody assays for evaluating candidate SARS

vaccines: Summary of a technical meeting 25–26 August 2005,
London, UK
Anjeanette Roberts a , John Wood b , Kanta Subbarao a , Morag Ferguson b ,
David Wood c , Thomas Cherian c,∗
a National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA
b National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK
c Department of Immunization, Vaccines and Biologicals, World Health Organization,

20 Avenue Appia, 1211 Geneva 27, Switzerland

Received 4 July 2006; accepted 5 July 2006

Available online 18 July 2006

Abstract

Severe acute respiratory syndrome (SARS) emerged in the Guangdong province of China in late 2002 and spread to 29 countries. By
the end of the outbreak in July 2003, the CDC and WHO reported 8437 cases with a 9.6% case fatality rate. The disease was caused by
a previously unrecognized coronavirus, SARS-CoV. Drawing on experience with animal coronavirus vaccines, several vaccine candidates
have been developed and evaluated in pre-clinical trials. Available data suggest that vaccines should be based on the the 180 kDa viral spike
protein, S, the only significant neutralization antigen capable of inducing protective immune responses in animals. In the absence of clinical
cases of SARS, candidate vaccines should be evaluated for efficacy in animal models, and although it is uncertain whether the United States
Food and Drug Administration’s “animal rule” would apply to licensure of a SARS vaccine, it is important to develop standardized animal
models and immunological assays in preparation for this eventuality. This report summarizes the recommendations from a WHO Technical
Meeting on Animal Models and Antibody Assays for Evaluating Candidate SARS Vaccines held on 25–26 August 2005 in South Mimms, UK,
provides guidance on the use of animal models, and outlines the steps to develop standard reagents and assays for immunological evaluation
of candidate SARS vaccines.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: SARS virus; Animal models; Vaccines; Guidelines

1. Introduction quarantine and isolation procedures, culling of wild animals

in live exotic animal markets and international collaboration
Severe acute respiratory syndrome (SARS) is a severe under the coordination of WHO [3]. By the end of the out-
respiratory illness caused by the SARS coronavirus (SARS- break, the CDC and WHO reported 8098 cases with a 9.6%
CoV) [1]. The disease emerged in the Guangdong province case fatality rate [4]. Only sporadic cases have been reported
of China in late 2002 [2] and spread to 29 countries mostly since then, mainly linked to laboratory exposure.
within Asia, although Europe and North America were also A novel virus was isolated in Vero cells from the respi-
affected, notably Toronto, Canada. The epidemic was finally ratory secretions from a patient with SARS [5,6]. Sequence
controlled by July 2003 through strict implementation of analysis showed that it was a previously unrecognized coro-
navirus, SARS-CoV [7–9]. Serological and genetic evidence
∗ Corresponding author. Tel.: +41 22 791 4460; fax: +41 22 791 41 93. supports a zoonotic origin of SARS-CoV [10,11]. Animal
E-mail address: cheriant@who.int (T. Cherian). traders working with masked palm civets in China had high

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2006.07.009
 A. Roberts et al. / Vaccine 24 (2006) 7056–7065 7057

prevalence for SARS-CoV antibody, although they had no The United States FDA “animal rule” states that, when effi-
history of SARS-like disease. SARS-CoV-like viruses that cacy studies in humans are not feasible, vaccines may be
were isolated from civets and raccoon dogs had more than approved based on animal data alone, provided the patho-
99% homology with human SARS-CoV, with major differ- physiological mechanism of the disease is reasonably well-
ences found in ORF8, whose deletion has been suggested understood as is its prevention or reduction by the vaccine.
to represent a sign of adaptation to humans [12]. Only four Moreover, the protective effect of the vaccine should be
amino acid residues in the receptor glycoprotein ACE2- demonstrated in more than one animal species expected to
binding domain of the viral spike protein differ between the react with a response predictive for humans. The endpoint of
human epidemic SARS-CoV strains and civet strains, but animal studies should be clearly related to the desired benefit
they cause more than a 1000-fold difference in binding affin- in humans (i.e. enhancement of survival or reduction in major
ity to the ACE2 molecule [13,14]. Although a high prevalence morbidity), and the data generated should allow selection of
of SARS-like coronaviruses were found in Chinese horseshoe an effective dose in humans. At the present time it is uncer-
bats [15,16], their great genetic diversity makes it difficult to tain whether the “animal rule” would apply to licensure of a
identify which one might be the ancestor of SARS-CoV and SARS vaccine. However, it is important to develop standard-
to decide with certainty whether bats indeed are the animal ized animal models and immunological assays in preparation
reservoir of the virus. for this eventuality.
SARS-CoV infection exhibits a wide clinical course char- Scientists at the WHO Technical Meeting on Animal Mod-
acterized mostly by fever, dyspnea, lymphopenia and lower els and Antibody Assays for Evaluating Candidate SARS
respiratory infection, often with concurrent gastrointestinal Vaccines held on 25–26 August 2005 in South Mimms, UK,
symptoms including diarrhea [17,18]. Pathology in SARS discussed many aspects of research pertaining to the use of
patients has been associated with diffuse alveolar damage, animal models in vaccine development including available
epithelial cell proliferation and multinucleated giant cell animal models, suitability of the various models, correlates of
infiltrates of epithelial or macrophage origin, suggestive of protection, critical components of potential vaccines, and the
syncytium-like formation in the lung. The virus can be recov- potential for disease enhancement in vaccinated animals fol-
ered from peripheral blood mononuclear cells, respiratory lowing exposure to SARS-CoV. In addition, standardization
secretions, stools, urine and even sweat (for a review, see of antibody assays and the establishment of a WHO Interna-
[19]). SARS vaccine development efforts were initiated very tional Standard for SARS-CoV antibody were also discussed.
rapidly after the identification of the etiologic agent, even This report endeavors to summarize the recommendations
though the immune correlates of protection were not known. from this meeting, based on consensus agreement. Recom-
Research efforts to identify protective antigens and to develop mendations for use of each animal model are given in Section
animal models were undertaken in parallel with efforts to 2 below. Correlates of protection, an overview of vaccine
develop candidate vaccines [20], drawing on experience with development, and observations pertaining to potential dis-
animal coronavirus vaccines and using several vaccine strate- ease enhancement are summarized in the following Sections
gies, including inactivated virus vaccines, purified subunit 3–6.
vaccines, plasmid DNA and viral vector-based vaccines as
well as virus-like particles. Much effort has been made to
identify appropriate animal models for SARS-CoV replica- 2. Animal models for evaluation of SARS-CoV
tion and pathogenesis. Several research groups have shown vaccines and antiviral treatments
that mice [21,22], ferrets [23], hamsters [24] and nonhu-
man primates [25–30] support replication of SARS-CoV with 2.1. General considerations
varying degrees of associated disease.
These animal models were used for the evaluation of can- In selecting animal models for vaccine evaluation, it is
didate vaccines, and the common conclusion that has emerged important to remember the principle underlying the so called
from the evaluation of several vaccines is that the 180 kDa “animal rule”, where data from more than one animal species
viral spike protein, S, is the only significant neutralization is often required: each animal species should contribute
antigen [31–34] and the only one to elicit protective immu- something different to our understanding of disease and pro-
nity in animal models [21,35–40]. The S protein can be tection. At this time, no single model offers a direct repro-
divided into two domains by analogy with other coronavirus duction of what was seen in humans with SARS. Pathology
spike proteins: an N-terminal S1 domain, which contains the (including pneumonitis, alveolar edema, and diffuse alveolar
receptor-binding site and neutralization epitopes and a C- damage (DAD)) in humans is probably the most difficult ele-
terminal S2 domain which forms the membrane-anchored ment to reproduce in an animal model. Attention also should
stalk region and contains a putative fusion peptide followed be given to the reduction of viral shedding because this would
by two heptad repeats predicted to form a six-helix coiled-coil likely correlate with decreased risk of further spread of the
bundle [41]. disease among humans. In using animal models to study
In the absence of clinical cases of SARS, candidate vac- aspects of SARS-CoV infection, it must be emphasized that
cines will have to be evaluated for efficacy in animal models. the kinetics of viral replication and appearance and resolu-
7058 A. Roberts et al. / Vaccine 24 (2006) 7056–7065

tion of pathological findings are much more rapid in animal mouse-adapted SARS-CoV strain MA-15. These were inoc-
models than in humans. ulated by the intranasal (IN) route under light anesthesia
Whichever animal model is employed, special considera- [21,42], using a dose of 105 TCID50 in 50 ␮L/mouse. Critical
tion should be given to the presence of co-existing pathogens, time points for specimen collections are days 2 (peak titer)
the age of the animals and the route(s) of infection. A suf- and 5 post-infection (p.i.) for quantitative virology, days 3
ficient number of time points and large enough number of and 5 p.i. for the study of interstitial pneumonitis and DAD
animals should be used to allow statistical evaluation. The in aged mice (inflate lungs with 10% neutral-buffered forma-
strain of SARS-CoV used also could be of importance. lin), and day 9 for resolution.
It should be emphasized that different species may prove
useful for studying different aspects of SARS-CoV. Whereas 2.2.2. Hamsters
vaccines or antivirals may be addressed in many models, Golden Syrian hamsters are an excellent animal model
pathogenesis is best evaluated in those animal models for because they demonstrate high levels of SARS-CoV repli-
which immunological tools and reagents are available for cation and develop pneumonitis. Hamsters are suitable for
detailed analysis of the immune response to the vaccine. This vaccine efficacy, immunoprophylaxis and treatment studies
includes inbred mice and rhesus and cynomolgus macaques. [24]. In contrast with BALB/c mice, in which the virus is
It may actually be worthwhile to enhance the virulence of detected only in the respiratory tract and is cleared by day
a SARS-CoV isolate by serial passages in an animal model 5 p.i., hamsters demonstrate a longer duration of viral shed-
to produce a challenge virus stock for vaccine studies that ding from the upper respiratory tract, some transient viremia,
would elicit more reproducible disease in the animals. If a spread of the virus to liver and spleen and, most significantly,
highly virulent host-adapted virus were to become available, inflammation of respiratory tissues [21,24].
such as a mouse-adapted or a monkey-adapted SARS-CoV SPF animals older than 5 weeks of age should be used
strain, demonstration of the capacity of vaccines to protect in sufficient number for statistical analysis and study design
against challenge with these more virulent strains would pro- should include mock-infected animals. The animals can be
vide an almost ideal animal model. housed in pairs or by three if the experiment is to last less than
Different models may also need to be employed to eval- 4–6 weeks. Reserve space should be available to separate the
uate pathogenesis versus immunogenicity. For pathogenesis animals in case of fights. Males and littermates tend to fight
studies in animal models, mortality is not required as a read- less.
out. It would be ideal to develop animal models with com- Virus strains that have been tested in hamsters are Urbani,
parable levels of mortality to that seen in humans (∼10% Frankfurt and HKU-39849. Virus should be administered
overall), including the increased mortality at increased age by the IN route under light anesthesia, using a dose of
(∼50% > age 60). The optimal result would be to demon- 103 TCID50 in 100 ␮L/hamster.
strate efficacy of vaccines or antivirals in SARS-CoV animal As an outcome of efficacy studies, quantitative virology
models that mimic human morbidity and mortality and that should be preferred over quantitative PCR. For pathology
show protection without vaccine-associated immunopathol- studies, one can grade pathology as none, mild, moderate
ogy. or severe as per Roberts et al. [24]. Critical time points for
specimen collection are days 2 or 3 p.i. (peak viral titer) and
2.2. Recommended animal models 7 (clearance) for quantitative virology studies; and days 5 p.i.
(consolidation and pneumonitis) and 14 or 21 (resolution) for
2.2.1. Mice pathology studies (lung).
Inbred mouse strains (BALB/c, C57BL/6, 129SvEv,
STAT1−/−) support SARS-CoV replication and can develop 2.2.3. Ferrets
pneumonitis (129S), but pneumonia and clinical symptoms There is evidence from one study that ferrets support
are only observed in older BALB/c mice [24]. The mouse SARS-CoV replication and develop pulmonary lesions [23]
model is suitable for immunogenicity and efficacy studies of but according to another study, the animals remain asymp-
vaccines. tomatic, in the presence of SARS-CoV replication [38]. In
Prolonged viral replication, dissemination of virus to view of these conflicting data, the ferret model needs to be
liver and spleen and accompanying pathology are seen in further studied to determine its optimal utility for vaccine
STAT1−/− mice; these mice, therefore, also are suitable for efficacy and immune prophylaxis studies. Additional stud-
studies of pathogenesis and evaluation of antiviral drugs. Spe- ies are needed to define the extent of biological variability
cific pathogen-free (SPF) animals must be used. Their age can of the model and the possible role of co-pathogens that may
be either 4–8 weeks or over 12 months and should be speci- contribute to the variability observed between different lab-
fied. The number of animals included must be sufficient for oratories.
statistical analysis, and should include mock-infected con- Animals aged 6 months or older should be used. Although
trols. not well documented, more consistent viral replication,
A variety of SARS-CoV strains has been tested in mice, pathology and clinical symptoms seem to be observed in
including Urbani, Frankfurt, HKU-39849, Tor-2, and the older animals. The animals should be screened for viral co-
 A. Roberts et al. / Vaccine 24 (2006) 7056–7065 7059

pathogens: Aleutian mink disease, respiratory viruses, hep- in cynomolgus macaques and AGMs, and later than day 4
atitis viruses, and others. The route of inoculation may be p.i. for rhesus macaques of Chinese origin. For collection
IN or IT, but not IV. The dose of virus (strains Tor-2, HKU- of tissues for histopathological analyses, days 2–4 p.i. are
39849) sufficient to ensure reproducibility of infection in all optimal for cynomolgus macaques and AGMs, and later than
animals is likely to be 105 pfu or more/ferret. day 4 p.i. for rhesus macaques. Due to limitations of immuno-
Again, quantitative virology is preferred over qRT-PCR. logical reagents (including microarray assays now available),
For pathology studies, the same recommendations apply as research may be limited to rhesus and cynomolgus macaques.
for nonhuman primate studies (see below): slides should be
shared between pathologists to develop a scoring system. 2.2.5. Other animal models: civets, rats, guinea pigs,
Regarding specimen collection of respiratory tissues, further wild voles
studies are needed to establish how much variation occurs in Data on other animal models are insufficient for consider-
samples from different lobes of the lungs. Critical time points ation for use in SARS-CoV vaccine and antiviral evaluations
are day 3 p.i. for quantitative virology and days 4–5 p.i. for [43–45]. Any additional model other than the four listed
pathology studies (pneumonitis). above (Section 2.2.1 to Section 2.2.4) would require thor-
ough characterization including viral replication data and
2.2.4. Nonhuman primates (NHP) histopathological analysis of SARS-CoV-infected and mock-
NHPs support SARS-CoV replication and develop pneu- infected animals of the same age and gender. Viral replication
monitis with a variable degree of clinical symptoms depend- and histopathological data in any new animal model should
ing upon the species employed. No single NHP species is be reminiscent of at least some aspect of SARS in humans.
preferred at this time. The number of animals in a given
study needs to be large enough to account for animal-to-
animal variability: a sample of 4 or 5 animals is not sufficient. 3. Correlates of protection
In view of the cost of the experiments, challenge studies
should be limited to those vaccine candidates that are most Although all the correlates of protection from SARS asso-
promising, using larger sample sizes (10–12 animals/group) ciated disease have not been identified in human infections,
and avoiding animals with free-range periods in life if pos- neutralizing antibodies are present in convalescent human
sible. Immunological responses are best studied in species serum. Antibodies to SARS-CoV spike (S) protein have been
for which microarrays and reagents for identifying immune shown to prevent virus entry and neutralize virus infectivity in
components are available, such as rhesus or cynomolgus vitro [32,46]. Prophylactically administered monoclonal anti-
macaques. However, the limited viral replication observed in bodies and passively transferred SARS-CoV hyper-immune
cynomolgus macaques might be a disadvantage in selecting sera have been shown to prevent SARS-CoV infection and
this species for studies. Other recommended NHP species are associated disease following SARS-CoV challenge of naive
the common marmoset and African green monkeys (AGMs, mice and hamsters [21,34,47–49]. Monoclonal antibodies
Chlorocebus aethiops sabeus). The country of origin may administered therapeutically (i.e. post-infection) also have
play an important role and should be specified, e.g. Philip- been shown to limit viral replication and reduce associated
pines (cynomolgus macaques, Macaca fascicularis); China disease in hamsters [50].
(rhesus, Macaca mulatta); Brazil (marmosets, Callithrix jac- Although cell mediated immunity may have a protective
chus), etc. role in viral clearance or resolution of disease, work in animal
Prior to the experiment, the animals should be housed models shows that antibody alone is effective for preven-
indoor to limit exposure to potential co-pathogens. They tion and treatment of SARS. Thus, mice immunized with
should be screened for parasites (Strongyloides, Pneumonys- live-recombinant vaccines expressing the SARS-CoV spike
sus simicola (lung mites)) and for possible viral co-pathogens protein, using rabies virus [51], vesicular stomatitis virus
(retroviruses, respiratory viruses, adenoviruses). The SARS- (VSV) [52], adenovirus (Ad5) [27,53] or attenuated vaccinia
CoV strains tested in NHP models are HKU-39849 (cynos), virus MVA [36,38] as a vector, as well as mice immunized
PUMC (rhesus) and Urbani (marmosets and AGMs). These with DNA vaccines expressing the S gene [37,54], devel-
were inoculated by the respiratory route (IN, IT) at a dose of oped neutralizing antibodies to SARS-CoV and were pro-
106 pfu or more/NHP. tected against SARS-CoV challenge. Similar findings were
Here again, quantitative virology is preferred over qRT- reported after mucosal immunization of hamsters and AGMs
PCR. For pathology studies, it would be an obvious advantage using a bovine parainfluenza virus type 3 (BPIV3) vector
that laboratories share pathology slides for review by different expressing the SARS CoV S gene [33,39]. Several whole
pathologists in order to develop a scoring system. Speci- inactivated virus and recombinant protein candidate vaccines
mens of respiratory tissues should be collected, but further also have been developed and shown to elicit a neutralizing
studies are needed to establish how much variation occurs antibody response that provided protection against infec-
in samples from different lobes of the lungs, as was done tious challenge [55–60]. In addition, passively administered
and reported for African green monkeys (AGMs) [28]. Crit- sera from vaccinated animals prevented SARS-CoV infection
ical time points are days 2–4 p.i. for quantitative virology upon subsequent challenge of naı̈ve mice, demonstrating that
7060 A. Roberts et al. / Vaccine 24 (2006) 7056–7065

antibodies induced by these vaccines did confer protection viral entry, as observed in these in vitro studies, has not been
[37,52]. related to any known component of human disease or infec-
The neutralizing antibody titer that is necessary to achieve tion in vivo. However, given that SARS-CoV may replicate,
protection in humans exposed to SARS-CoV is, however, still albeit poorly, in human PBMCs [73,74], in vitro experiments
not known. looking for antibody-enhancement of SARS-CoV replication
It was recommended that when evaluating vaccine efficacy in human cells (e.g. macrophages and B-cells) should be per-
in future animal experiments, the challenge virus should be formed.
administered at two different time-points, once when post- Several groups have studied SARS-CoV infection in ani-
immunization neutralizing antibody titers are high, and later mals in the presence of neutralizing and sub-neutralizing
when neutralizing antibody titers have waned or are low. It levels of SARS-CoV anti-sera or anti SARS-CoV S-protein
also was suggested that viral titers and pathology should be monoclonal antibodies, but no evidence of enhanced res-
evaluated at two different time points. Specific times points piratory disease has been observed. However, foci of hep-
for sample collection are given for each animal model in atic necrosis were noted following SARS-CoV challenge in
Section 2 above. MVA-SARS-S immunized ferrets [38]. Although these find-
ings are worrisome, several questions were raised regarding
the significance of the observation. The MVA-SARS-S vac-
4. Disease enhancement cine used in these experiments was poorly immunogenic
in the ferrets. The question of whether there could have
Previous observations of disease enhancement have been been any co-pathogen in the animals was raised. It also
reported for human viral pathogens and shown to be due to would be important to know if the observed phenomenon
antibody-mediated enhancement of virus entry (for reviews depends on the MVA vector or on the animal model. It was
see [61,62]). Enhanced disease and mortality have been strongly urged, therefore, that the experiment be repeated in
observed in kittens immunized against or infected with a type- ferrets. Additional experiments, in nonhuman primates and
I coronavirus, feline infectious peritonitis virus (FIPV), when hamsters, looking for evidence of enhanced respiratory and
subsequently exposed to FIPV infection [63–65]. Aggra- hepatic diseases upon vaccination and challenge were also
vated FIP is apparently a result of enhanced viral entry into encouraged.
macrophages mediated by sub-neutralizing antibody levels
[66]. Children vaccinated with inactivated respiratory syn-
cytial virus (RSV) vaccines developed serious disease on 5. Standardization of immunological assays—a
subsequent exposure to RSV [67–69]. Individuals exposed to proposed International Standard for SARS-CoV
one of the four serotypes of Dengue virus developed severe antibody
disease when subsequently infected with a second, different
serotype [70,71]. Enhanced disease following RSV vaccine Several candidate SARS vaccines that are at various stages
or dengue infection occur by different mechanisms than FIPV. of pre-clinical and clinical development are being developed
In view of such examples of enhanced disease following worldwide. In China alone, three companies have been given
infection in a vaccinated host, there has been heightened regulatory approval for the clinical evaluation of a candidate
concern that a similar phenomenon could occur with SARS- SARS vaccine. It is important, therefore, to be able to com-
CoV vaccines. It was highly recommended, therefore, that pare data from each of the candidate vaccines, which, in turn,
known mechanisms of disease enhancement observed with requires international standardization of the immunological
other viruses and especially with other coronaviruses should assays used for the evaluation of these vaccines.
be examined in SARS-CoV infections, especially in vacci- The accepted method of international standardization is
nated animals. Although none of the studies to date have to employ a WHO International Standard (IS), which allows
shown enhanced respiratory disease following SARS-CoV comparison of results from different laboratories [75]. This is
challenge in previously immunized animals, further studies essential for establishing international requirements for vac-
in this area are warranted in view of some of the available in cines, diagnostics or therapeutics. An IS is prepared from
vitro data. material bearing a close resemblance to the samples being
Antibodies against human SARS-CoV isolates were assayed; the material is distributed in glass ampoules with
shown to enhance the entry of pseudo-typed viruses express- high precision and reproducibility and then freeze dried. It is
ing the civet SARS-like CoV-spike protein into a human renal important that a sufficient number of ampoules (2000–3000)
adenocarcinoma cell line (786-O). Enhancement was only be prepared so as to provide for about 10 years of use, and that
demonstrated at the level of entry, but not of replication [72]. the activity of the contents remain stable over this period. The
This phenomenon was seen with pseudo-typed lentiviruses process of establishing a WHO IS involves an international
expressing SARS-CoV spike protein of civet sequence speci- collaborative study, in which the candidate IS is compared
ficity, but not with pseudo-typed viruses expressing spike pro- with other samples. If the results of the tests are suitable,
teins of human SARS-CoV isolates. It also was not observed the candidate IS assigned a provisional arbitrary unitage, a
with human isolates of SARS-CoV. The role of enhanced report is distributed for approval by study participants and
 A. Roberts et al. / Vaccine 24 (2006) 7056–7065 7061

for eventual approval by the WHO Expert Committee on tion of plasma, quality control testing and standardisation of
Biological Standardization (ECBS). The preparation, storage assays. Three Chinese manufacturers have been licensed for
and distribution of over 90% of IS have been undertaken by preparation of SARS immunoglobulin. The source material
the National Institute for Biological Standards and Control was plasma from convalescent patients at more than 28 days
(NIBSC) at South Mimms (UK), which is a WHO Laboratory after infection. All were in good health and their plasma tested
for Biological Standards. negative for blood-borne agents. Plasma samples were pro-
NIBSC has developed in the past several WHO ISs for cessed by a combination of cold ethanol fractionation and
calibration of the antibody response against virus vaccines, ultrafiltration. In September 2003, three lots of IgG were
including ISs for antibodies against Dengue, Hepatitis A produced and assayed by nationally-agreed procedures. The
virus (HAV), [76] Hepatitis B virus (HBV), Measles, [77] stock of immunoglobulin currently available is sufficient to
Polio, Rabies, [78] Rubella, and Smallpox. A candidate treat 100 patients. Monoclonal antibodies have not yet been
standard against Human Papilloma virus-16 (HPV-16) is considered for treatment purposes in China.
under evaluation. The corresponding IS’s were used for The importance of assessing immunogenicity of candidate
a variety of antibody assays including virus neutralisation SARS-CoV vaccines using VN assays is well acknowledged,
(VN), haemagglutination-inhibition, single radial diffusion, but the variety of VN tests in use is a significant problem
enzyme-linked immunoassay and radio-immunoassay. Anti- since there is at this time no consensus on the most sensi-
body ISs are most useful in epidemiological studies and tive, specific, and reproducible assay system. It is therefore
in clinical trials. Their use allows correlates of immunity desirable to establish an antibody IS to serve as a basis of
and potency requirements of prophylactic and therapeutic comparison in all VN assays. The most important activ-
products to be expressed in International Units (IUs). Data ity at this time is to obtain a suitable source of antibody.
from several collaborative studies demonstrate that use of an A number of options can be considered, such as convales-
IS generally reduces the level of variability between assay cent human sera, post-immunization human sera, monoclonal
results. antibodies or hyperimmune animal sera. As an example, the
However, there may be problems in using ISs due to the availability of a suitable source of serum from convales-
complex array of antibody populations in each serum and the cent patients in Hong Kong needs to be explored, although
different sensitivity of different assay systems. Examples of antibody levels in these individuals are probably quite low
potential problems can be found in HBV and Parvovirus B19 by now.
studies [79], which showed that different assay kits gave dif- It also would be important that other assays than VN be
ferent results even when the IS was included in the assays. included in the collaborative study, and that the impact of
Another issue is the degree of antigenic homology between SARS-CoV strain variation be examined by using different
the viral antigen used for the preparation of the IS and the SARS-CoV strains and/or sera with different specificities.
virus used in the assays. In a JEV collaborative study, a can- The Centralized Facility for AIDS Reagents (CFAR),
didate antibody IS, which had been prepared from the sera which is based at NIBSC, could be a suitable model
of vaccinees immunized with an inactivated vaccine that was for a SARS-CoV repository [80]. The CFAR was estab-
antigenically different from some of the viruses used in VN lished in 1989 to support AIDS vaccine research and it
assays, demonstrated that the response to at least some inac- is now EU-funded [81]. There are currently 2000 reagents
tivated vaccine is strain specific and the candidate IS was available including peptides, recombinant proteins, human
consequently not established by the WHO ECBS. Whether a sera/plasma, monoclonal and polyclonal antibodies, expres-
panel of monoclonal antibodies to SARS-CoV could be used sion systems, cDNA clones and viruses. A comparison can be
to prepare an IS is an attractive alternative which should be drawn between SARS-CoV and HIV VN assays. Currently,
explored. there are several different HIV neutralization assays formats
Significant progress with standardisation of SARS-CoV under consideration and a lack of agreement on the most suit-
antibody assays had been made in China with the develop- able assay. The CFAR is supporting a joint WHO/EU project
ment of a national antibody standard. In order to develop the (NeuNET) to evaluate and standardize HIV VN assays in an
national standard, sera were collected from 20 convalescent international collaborative study.
SARS patients who were found to have SARS-CoV VN anti- In the USA, a SARS-CoV repository has been established
body titers ranging from undetectable to 1:203. One serum on behalf of the American Type Culture Collection (ATCC)
sample was selected for further evaluation based on cross- in order to meet the needs of biodefence and the threat of
reactivity with four SARS-CoV strains and on Western blot emerging infections [82]. The type of reagents stored includes
analysis. This serum was freeze dried in 0.5 mL aliquots and viruses, peptide arrays, monoclonal antibodies and proteins.
was then assessed for stability by VN assays. The Chinese It is hoped that an active collaboration can be established
standard was assigned a VN titer of 52.7 with 95% confidence between NIAID and NIBSC in order to meet the expanding
limits of 47.6–62.2. needs of the SARS research community.
A further important development in China was the prepa- Based on the discussion at the meeting, the following rec-
ration of human immunoglobulin for treatment of SARS ommendations were made with respect to standardization of
patients. National guidelines have been prepared for collec- the immunological assays for SARS vaccine evaluation:
7062 A. Roberts et al. / Vaccine 24 (2006) 7056–7065

1. A WHO repository for SARS-CoV reagents ought to be cacy against different strains of SARS-CoV. The implications
developed. Collaboration between NIAID and NIBSC is of the sequence heterogeneity among SARS-CoV strains are
recommended to achieve this goal. difficult to test at this time because the most divergent strains
2. Consensus must be reached for the reagents to be given (civet SARS-like viruses) have not been recovered in culture.
priority in the repository. Validation and international standardization of immuno-
3. An International Standard for SARS-CoV antibody is logical assays for the evaluation of candidate SARS vac-
needed. cines are essential to compare data across different trials.
4. The most suitable source of antibody for the IS is con- This requires the establishment of International Standards
valescent human sera, but post-vaccination human sera for SARS-CoV antibody and a repository for SARS-CoV
could also be used. reagents, with an international collaborative study to vali-
5. A protocol for an international collaborative study aimed date the ISs. The establishment of the repository by WHO in
at validating the IS should be developed and distributed collaboration with NIBSC and NIAID was recommended.
to prospective participants.
6. Collaborative study participants should be asked about
their assay capabilities, e.g. number of sera, virus strains List of meeting participants
handled, etc. . .
7. The proposed IS collaborative study should include a core
Dr. Larry J. Anderson, Chief, Respiratory and Enteric Viruses Branch,
set of antibody preparations to be distributed and assayed Centers for Disease Control and Prevention, USA
in each laboratory (e.g. monoclonal antibodies, animal Dr. Tracey Baas, Katze Laboratory, University of Washington, USA
sera, other human sera). *Dr. Lorne Allan Babiuk (could not attend), Vaccine & Infectious Disease
8. Tests should be conducted using the same strain of SARS- Organization (VIDO), University of Saskatchewan, Canada
Professor Jianshi Bai, Deputy Director, Division of Blood Products,
CoV in each laboratory, but the different genetic lineages
National Institute for the Control of Pharmaceutical and Biological
of SARS-CoV should also be represented in the study. Products, People’s Republic of China
*Dr. Ralph Baric (could not attend), University of North Carolina, USA
Dr. Frederick J. Cassels, SARS Program Officer,
6. Biosafety issues RDB/DMID/NIAID/NIH/DHHS, USA
Dr. Thomas Cherian, Department of Immunization, Vaccines and
Biologicals (IVB), World Health Organization, Switzerland
Biosafety issues associated with SARS-CoV vaccine Dr. Markus Czub, Canadian Science Centre for Human and Animal
development stem from the reports of laboratory-acquired Health, Canada
infections in China. Sanofi Pasteur has adopted BSL 4 prac- Dr. Rose Das, National Institute for Biological Standards and Control,
tices and BSL 3 equipment (e.g. Class 2 or 3 microbiological United Kingdom
Professor Guanmu Dong, Director, First Division of Viral Vaccines,
safety cabinets with respiratory protective equipment) for the
National Institute for the Control of Pharmaceutical & Biological
preparation of SARS-CoV vaccines. Of note is the fact that Products, People’s Republic of China
SARS-CoV appears to be quite resistant to normal methods Dr. Morag Ferguson, Senior Virologist, National Institute of Biological
of virus decontamination (JF Saluzzo, personal communica- Standards and Control, United Kingdom
tion). WHO has developed guidance, both general [83], and *Dr. Reinhard Glueck (could not attend), Berna Biotech AG, Switzerland
Dr. Bart Haagmans, Institute of Virology, Erasmus Medical Centre, The
specific for handling SARS specimens [84].
Netherlands
*Dr. Wei He (could not attend), Deputy Director, Peking Union Medical
College, People’s Republic of China
7. Conclusions *Dr. Stephen L. Hoffmann (could not attend), Chief Executive and
Scientific Officer, Sanaria Inc., USA
Dr. Harvey Holmes, National Institute for Biological Standards and
The rapid success in the development of immunogenic
Control (NIBSC), United Kingdom
and protective vaccines against SARS using a variety of plat- Dr. Katja Hoschler, Clinical Scientist, Health Protection Agency, United
forms is encouraging, but should be tempered with concerns Kingdom
about the possibility of enhanced disease following exposure Dr. Koji Ishii, Department of Virology, National Institute of Infectious
in vaccinated individuals [85]. Concerns mainly stem from Diseases, Japan
*Dr. Steven Jones (could not attend), Special Pathogens Program, Health
reports of enhanced disease in FIPV-immunized or -infected
Canada, Canada
kittens [63,66], from observations that antibodies elicited Dr. Colleen Jonsson, Homeland Security and Infectious Diseases, Southern
against certain coronaviruses mediate antibody-dependent Research Institute, USA
enhancement of viral entry [65], and from the observation *Dr. Marie-Paule Kieny (could not attend), Director, Initiative for Vaccine
of inflammatory foci in liver tissue following SARS-CoV Research, World Health Organization, Geneva, Switzerland
*Dr. Otfried Kistner (could not attend), Director Virology, Baxter
challenge in MVA-SARS-S vaccinated ferrets [38].
BioScience, Austria
Candidate SARS vaccines will need to be evaluated in Dr. Diane Major, National Institute for Biological Standards and Control,
more than one animal model. They also will need to be thor- United Kingdom
oughly evaluated for the duration of the antibody response Dr. Keith Mansfield, Chair of the Division of Primate Resources, New
they induce, as well as for the breadth of their protective effi- England Primate Research Center (NEPRC), USA
 A. Roberts et al. / Vaccine 24 (2006) 7056–7065 7063

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