Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans.

Abstract

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.

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Development of a Mouse Monoclonal Antibody Cocktail
for Post-exposure Rabies Prophylaxis in Humans
Thomas Mu ¨ller1, Bernhard Dietzschold2, Hildegund Ertl3, Anthony R. Fooks4, Conrad Freuling1, Christine
Fehlner-Gardiner5, Jeannette Kliemt1, Francois X. Meslin6, Charles E. Rupprecht7, Noe ¨l Tordo8,
Alexander I. Wanderler5, Marie Paule Kieny9*
1WHO Collaborating Centre for Rabies Surveillance and Research, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Wusterhausen, Germany,
2WHO Collaborating Centre for Neurovirology, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of
America, 3WHO Collaborating Centre for Reference and Research on Rabies, Wistar Institute, Philadelphia, Pennsylvania, United States of America, 4WHO Collaborating
Centre for the Characterization of Rabies and Rabies-related Viruses, Veterinary Laboratories Agency, Department of Virology, New Haw, Addlestone, Surrey, United
Kingdom, 5WHO Collaborating Centre for Rabies Control, Pathogenesis and Epidemiology in Carnivores, Canadian Food Inspection Agency (CFIA) Centre of Expertise for
Rabies, Ottawa, Ontario, Canada, 6Neglected Zoonotic Diseases (NZD), Department of Neglected Tropical Diseases (NTD), Cluster HIV/AIDS, Malaria, Tuberculosis and
Neglected Tropical Diseases (HTM), World Health Organization, Geneva, Switzerland, 7WHO Collaborating Centre for Reference and Research on Rabies, Rabies Section,
Division of Viral and Rickettsial Diseases, Viral and Rickettsial Zoonoses Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta,
Georgia, United States of America, 8Unit Antiviral Strategy, CNRS URA-3015, Institut Pasteur, Rabies Unit, Paris, France, 9Initiative for Vaccine Research, Vaccines &
Biologicals, Health Technology & Pharmaceuticals, World Health Organization, Geneva, Switzerland
Abstract
As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the
limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive
immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially
cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this
study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at
the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14,
1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as
biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each
hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III
on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique
MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions.
Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of
targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails
neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb
cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel
combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less
expensive alternative biological agent for use in PEP and prevention of rabies in humans.
Citation: Mu ¨ller T, Dietzschold B, Ertl H, Fooks AR, Freuling C, et al. (2009) Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies
Prophylaxis in Humans. PLoS Negl Trop Dis 3(11): e542. doi:10.1371/journal.pntd.0000542
Editor: Jakob Zinsstag, Swiss Tropical Institute, Switzerland
Received June 24, 2009; Accepted October 6, 2009; Published November 3, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: ARF was funded by the UK Department for Environment, Food and Rural Affairs (Defra grants SEV3500 and FT5091). TM and CF were funded by the
Federal Ministry of Health and the Federal Ministry of Food, Agriculture and Consumer Protection, Germany. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Kienym@who.int
Introduction
Rabies is an acute viral encephalomyelitis in humans and other
warm-blooded vertebrates, caused by a member of the genus
Lyssavirus of the Rhabdoviridae family. Within the genus, seven
genotypes (gts) have been delineated and the classification for
another four recently found viruses within the genus is still pending.
Lyssavirus Gts have been further segregated into phylogroups on
the basis of their glycoprotein gene sequence, and the pathogenicity
and immunogenicityof the virus.Theprototypevirusofthe genusis
rabies virus (RABV; gt 1), which along with Duvenhage virus
(DUVV; gt 4), European bat lyssavirus type-1 and -2 (EBLV-1
and -2; gts 5 and 6, respectively), belongs to phylogroup I [1]. The
unclassified lyssaviruses Aravan virus (ARAV), Khujand virus
(KHUV) and Irkut virus (IRKV) also cluster with this group [2].
The African gts, Lagos bat virus (LBV; gt 2) and Mokola virus
(MOKV; gt 3) were assigned to phylogroup II [1]. Studies have
shown that West Caucasian Bat virus (WCBV) is the most divergent
member of the genus and may not belong to either phylogroup I or
II but rather represents a new phylogroup III [2,3].
Classical rabies caused by the prototype RABV is the most
important public health problem world-wide. Only certain countries
e.g. the United Kingdom, New Zealand, the state of Hawaii (USA),
Australia and Antarctica and parts of Western Europe, are currently
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free of the virus, either historically or through successful rabies
elimination programs. The epidemiology of this enzootic disease in
rabies endemic countries is characterized by the principal reservoir
host species in which the virus circulates. Two broad circulation
patterns are recognized: sylvatic rabies (involving wildlife in both
carnivora and chiroptera orders) and canine rabies, which represents the
heaviest burden on human health. The occurrence of these two
circulation patterns follows a general geographic and socio-economic
pattern [4]. Canine rabies causes an estimated 55,000 human deaths
each year, especially in Asia and Africa, although the true burden of
the disease is unknown due to underreporting and poor surveillance
systems in many areas of the world [5–7]. It has been estimated that
half of the world’s population live in a canine rabies-endemic area
[8]. Although the most efficient way of preventing human rabies
cases is the control of the disease in the vector population by mass
dog vaccination combined with population control, such efforts have
not been taken systematically in large parts of Africa and Asia. Also
effective vaccines that protect humans against rabies are not
universally available throughout the world. The largest number of
fatalities is reported in under-privileged children principally those
under14yearsof age that livein the poorer countriesoftheworld.In
greater than 99% of cases, human death results from dog-bite injury
[8]. In the majority of cases, a category 3 exposure occurs, which
includes bites and/or contamination of mucous membranes with
saliva containing the virus. In a rabies infected area, a category 3
exposure should be treated immediately by wound treatment
(thorough washing) plus the administration of rabies post-exposure
prophylaxis (PEP) comprised of both rabies immunoglobulin (RIG)
for passive protection and rabies vaccine to induce circulating virus-
neutralizing antibodies(VNAs)[4].Evidencesuggests that when PEP
is administered in a timely manner RABV is cleared before it enters
the CNS [9]. The mode of protection is likely to be virus
neutralization by antibodies or antibody-mediated clearance of
virus-infected cells [10–12].
Currently, human and equine polyclonal anti-rabies immune
globulin (HRIG and ERIG, respectively) are used in passive
immunization. They are prepared from pooled sera taken from
hyper immunized humans or horses, respectively [13]. HRIG is
available in limited quantities on specific markets and is prohibitively
expensive (approximately US$250 per adult treatment) for most
rabies virus-exposed humans living in developing countries. The cost
is approximately five times that of purified horse serum. ERIG
although being a potent biological may show significant differences in
adverse reaction rates, reflecting differing manufacturing or purifica-
tion processes and protein content and therefore may lead to
complications such as serum sickness or anaphylactic shock [13].
However, when modern purified ERIG is used the prevalence of
anaphylaxisandmildserumsickness-likereactionsisverylow[14,15].
Other problems arising from the production of both HRIG and
ERIG include high manufacturing costs and the potential risk
contamination in human blood products including unknown
agents and pathogens [16]. In addition, animal protection groups
that are becoming more influential in developing countries are
trying to stop animal usage for production of antisera.
Thus, besides efforts to improve the supply with HRIG and
ERIG, accelerated research and development of alternative
products are required and essential for the future of global health
practices in the management of human rabies. Monoclonal
antibodies (MAbs) are particularly attractive alternatives for
HRIG and ERIG and could be a keystone in rabies prevention
as they seem to represent a revolution in clinical medicine [17].
Mabs have also been approved to treat cancer, inflammatory and
other infectious diseases and to prevent graft rejection [18,19].
Anti-RABV glycoprotein (G) MAbs are considered alternatives to
HRIG and ERIG because they would be safer products for use in
PEP preparations for humans [4].
Mouse and human MAbs (MoMAbs and HuMAbs) that
neutralize RABV have been produced by different groups of
investigators [10–12,20–26]. Both MAb types could form the basis
for viable alternative strategies for PEP in humans as they have
many advantages over HRIG and ERIG. Ideally, a collection of
MAbs capable of neutralizing all RABV strains relevant to human
rabies would be required [16]. Till now, despite the identification
of numerous potential HuMAbs for rabies PEP, only one HuMAb
cocktail comprising two HuMAbs has been developed, thoroughly
characterized both in vitro and in vivo and successfully clinically
tested in two phase I studies [27–29].
Although MoMAbs have been used extensively for antigenic
typing of RABV strains and their protective activity has been
demonstrated in certain animal models [10–12,25], a unique
MoMAb cocktail combination to replace HRIG or ERIG has not
yet been developed. The objective of this WHO co-ordinated
project has been to evaluate existing MoMAbs at WHO
Collaborating Centres for their capacity to neutralize a variety
of RABVs, principally canid strains. The most promising of these
were then tested in combinations of a minimum of two anti-G
MuMAbs, targeting distinct antigenic sites, to replace HRIG and
ERIG for human PEP against rabies. Here we report three unique
MoMAb cocktail combinations that would be suitable replace-
ments for HRIG and ERIG, based on the stringent criteria for
each of the selected MoMAbs concerning neutralizing potency,
binding specificity, and spectrum of neutralization of ‘street’
RABV isolates. The final characterisation of these three unique
MoMAb cocktail combinations was based on their cross-reactivity
against RABVs in vitro and efficacy in vivo, in protecting animals
against rabies.
Materials and Methods
Selection of MoMAbs and technical information
Panels of well-defined neutralizing anti-G MoMAbs available
from four WHO collaborating centres for rabies were screened for
Author Summary
Human mortality from endemic canine rabies is estimated
to be 55,000 deaths per year in Africa and Asia, yet rabies
remains a neglected disease throughout most of these
countries. More than 99% of human rabies cases are
caused by infections resulting from a dog-bite injury. In
the vast majority of human exposures to rabies, patients
require post-exposure prophylaxis (PEP), which includes
both passive (rabies immunoglobulin, RIG) and active
immunization (rabies vaccine). The number of victims
requiring PEP has increased exponentially in recent years,
and human and equine RIG (HRIG and ERIG) were not
sufficiently available in countries where canine rabies is
endemic. Rabies virus-neutralizing monoclonal antibodies
(MAbs) of mouse (Mo) origin have been identified as
promising alternatives to HRIG and ERIG. We have
developed and assessed both in vitro and in vivo unique
mouse monoclonal antibody (MoMAb) cocktails, which are
highly efficacious. Three novel combinations were shown
to have an equal or superior efficacy to HRIG and therefore
could be considered a potentially less expensive alterna-
tive for passive prophylactic use to prevent the develop-
ment of rabies in humans, particularly where needed most
in developing countries.
Anti-rabies Mouse Monoclonal Antibody Cocktail
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their suitability as potential candidate MoMAbs using the
following selection criteria:
1. Biological activity–They should exhibit (i) a minimum
neutralizing potency of 100 IU/ml of crude hybridoma
supernatant, (ii) a consistent production stability (loss of
MoMAb secretion should not exceed 10% up to 30 passages),
(iii) a broad spectrum of reactivity with regard to genotype I
(canid strains);
2. Binding specificity–They should target distinct, non-overlap-
ping epitopes (antigenic sites I–III) on the RABV G;
3. Immunoglobulin isotype (Ig)–They should be preferably of
isotype IgG1, 2a or 3;
4. History of hybridomas–There should be sufficient background
information on the relative risk of possible contamination with
transmissible spongiform encephalitis (TSE) agents and on the
regional sources of known batches of fetal calf serum used for
hybridoma growth.
Further technical information on the recommended culture
conditions for hybridomas, available nucleotide sequence of heavy
and light chain cDNAs, and intellectual property rights issues were
also established (Table 1). In one case, a candidate MoMAb had to
be excluded from further consideration because of intellectual
property ownership; however, the MoMAb was still included in
the study for comparative purposes.
The minimum neutralizing potency of the crude hybridoma
supernatant and the relative stability of antibody produced, in
terms of its virus-neutralizing activity was determined under both
serum-containing and serum-free conditions using the rapid
fluorescent focus inhibition (RFFIT) test and the fluorescent
antibody virus neutralization (FAVN) test as described [30,31].
The IgG isotype or subtype of the MuMAbs was determined by a
commercially available dipstick typing test kit (Serotec, Du ¨sseldorf,
Germany) or an in-house-developed Fluoricon assay. Briefly,
polystyrene beads (IDEXX, Westbrook, USA) were coated with
unlabelled Ig (IgM+IgG+IgA, H and L chains; Southern Biotech,
Birmingham, USA), incubated in Fluoricon assay plates (IDEXX)
with hybridoma supernatant, washed and then incubated with the
FITC-labelled isotype-specific goat-anti-mouse antibodies (South-
ern Biotech). Unbound, labelled antibody was washed off and then
the plates were read in a Fluorescence Concentration Analyzer
(FCA, IDEXX).
The binding specificity of MoMAb candidates, determined by
the localization of the potential binding site on the virus G, was
assessed using two different approaches, either by generating
MoMAb-specific escape mutants or in vitro cross-neutralisation
assays. To generate MoMAb-specific escape mutants, fixed RABV
Table 1. Available technical information for candidate MoMAbs.
History of hybridomas E559.9.141112-162-7-13M727-5-1M777-16-3
Mouse strain providing B-cells BALB/c miceBALB/c mice BALB/c mice BALB/c miceBALB/c mice
AntigenERA G protein ERA G proteinwhole ERA whole ERA, #167–169whole ERA, #167–169
Fusion partner (Year of fusion)P3-X63Ag8 (1979) 653 (1985) Sp2/0–Ag14 myeloma
(1983)
Sp2/0–Ag14 myeloma
(1994)
Sp2/0–Ag14 myeloma
(1994)
Reference [35][50] nono no
Number of cloning steps4 Not known344
Purity/homogeneity of cell line Not knownNot known Sub-cloned 2x, single IgG peak isotype as pure IgG 2a isotype as pure IgG 1
Origin of FCS used New ZealandUSA USA (GIBCO)USA (Sigma), Canada
(Wisent)
USA (Sigma), Canada
(Wisent)
Absence of adventitious agents Mycoplasma freen.d.Per WHO screening requestn.d.n.d.
Culture conditions
MediumIscove’s DMEM 1DMEM (modified)Iscove’s DMEM 2 HY-HT (10% FCS)HY-HT (10% FCS)
Cell concentration104–106
104–106
26105
66104236105
76104236105
Serum-free culture mediumCD HM or PFHM II protein-
free
Not tested tested but no specificationUltradoma-PFUltradoma-PF
Type of immunoglobulin
IgG subtytpe IgG 1 (ELISA)IgG 1 (ELISA)IgG 2b (ELISA) IgG2a (FCA)IgG 1 (FCA)
Heavy/light chains cDNAs Yes Yesnono no
Antigenic site recognized on G IIII cIII IIII
Method for determining epitope sequencingsequencing cross-neutralisationcross-neutralisationcross-neutralisation
Escape mutants
derivation SAD B19 CVS-11 not availablenot availableERA
aa substitutions in G aa 57 (Leu to Arg) aa 53 (Gly to Glu)aa 198 (Lys to Glu)
aa 217 (Lys to Glu)aa 286 (Ala to Thr)
Production yield
Yield in IU/ml (crude hybridoma) 62.5330–6022–32 11–32
Legend: aa–amino acid, CVS 11–Challenge virus standard 11, DMEM–Dulbeccos’ minimum essential medium, ELISA–enzyme linked immunosorbent assay, ERA–Evelyn
Rokitniki Abelseth SAD derived RABV strain, FCA–Flouricon-CA Assay, HB–hybridization medium, SAD–Street Alabama Dufferin strain of RABV. Media specification: Iscove’s
DMEM 1=Iscove’s modified DMEM+HAM F12 (1:1)+10% FCS; Iscove’s DMEM 2=Iscove’s modified DMEM+ITS+antibiotics/antimycotics+L-glutamine+5% FCS.
doi:10.1371/journal.pntd.0000542.t001
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strains (SAD, CVS, ERA) were propagated in BHK-21 cells in
serial passages, at a multiplicity of infection of 0.01, in the presence
of a MoMAb at a sufficiently low concentration to have no
neutralizing effect. Subsequent sequence analysis of the G gene of
derived virus escape mutants and multiple alignments were
undertaken as described [32]. MoMAbs that did not generate
any escape mutants were checked for their neutralizing potency
with escape mutants derived from other MoMAb candidates or
from MAb D1 (Institut Pasteur, Paris, France), which is a well-
characterised antibody with a previously identified binding
specificity to binding site III on the RABV G [26,33,34].
In vitro neutralization studies
In a preliminary study, MoMAbs, either purified or in the form
of crude cell culture supernatant, were tested in vitro for their broad
spectrum of reactivity. In a second experiment, based on the
selection criteria in terms of binding specificity and broad
spectrum cross-reactivity, blinded mini-cocktails comprising of
two MoMAbs targeting different epitopes, e.g. binding sites, on the
RABV G, were assessed further under the same conditions (see
below). Testing of the in-vitro broad spectrum cross-reactivity of the
selected MoMAb candidates as well as representatives of all the
known lyssavirus gts (gt 1–7, n=28) was undertaken in three
independent laboratories: Friedrich-Loeffler-Insitute (FLI, Ger-
many), Centers for Disease Control and Prevention (CDC, USA),
and Canadian Food Inspection Agency (CFIA, Canada), as
described [23,35]. Putative lyssavirus genotypes (Aravan virus–
ARAV, Khujand virus–KHUV, Irkut virus–IRKV, and West
Caucasian Bat virus–WCBV) were also assessed (n=4). The
principal focus was on canid strains of RABV (gt 1) (N=20) from
specific host species and geographical areas across the world [4].
Prior to testing, monolayers of murine neuroblastoma cells (NA
42/13) were infected with selected lyssavirus field strains, at a
multiplicity of infection of 0.1, for 1 h at 37uC in 5% CO2.
Subsequently, the virus inoculum was removed and fresh Minimal
Essential Medium (MEM) was added to the cells. Following
incubation for 72 h at 37uC in 5% CO2, cell culture supernatants
were collected and titrated on BHK-21 or MNA cells (BioWhi-
taker, Walkersville, USA). Up to three passages were undertaken
to obtain sufficient virus titres. Viruses were stored at 280uC until
further use. The neutralizing potency of the MoMAbs was
determined by RFFIT or FAVN using BHK-21 or MNA cells
infected with a constant amount of virus and varying amounts of
the MAb (endpoint titration) as described [23,36]. Briefly,
MoMAbs and/or blinded mini-cocktails were serially diluted
and incubated with 102or 104FFU/ml of selected lyssavirus
strains for 24 h. Subsequently, virus growth was detected by fixing
the cells with cold 75% acetone and then staining with a
fluorescein isothiocyanate (FITC)-labelled anti-rabies conjugate.
VNA titres were expressed as the reciprocal of the dilution at
which 50% of the wells showed complete neutralization of virus
growth. The titres were compared to those of an international
standard rabies immunoglobulin (SRIG, 2nd human rabies
immunoglobulin preparation, National Institute for Biological
Standards and Control, Potters Bar, UK) adjusted to 30 IU/ml
and converted into international units per ml (IU/ml).
Batch production and testing of candidate MoMAbs
under GLP conditions
A Master Cell Bank was prepared for each candidate
hybridoma by the National Institute for Biological Standards
and Control (NIBSC, UK). A vial corresponding to each of the
selected hybridomas was provided to a service manufacturer
(Apotech, Lausanne, Switzerland) contracted by WHO for
production of MuMAbs under Good Laboratory Practice (GLP)
conditions in small-scale cultures using culture media as
recommended (Table 1). Subsequently, MoMAbs were purified
by Protein A affinity chromatography essentially as described [16].
The purity of all MoMAb preparations was assessed by
electrophoresis through a 12.5% polyacrylamide gel under
reducing conditions (SDS-PAGE) and subsequent Coomassie blue
staining. Yields were expressed in protein mass (mg/L). VNA titres
(IU/L) of the purified MoMAbs were determined by RFFIT in
three independent assays using CSV-11 as a challenge virus as
described [37]. Unique standardized cocktail combinations
consisting of two purified MoMAbs of equal concentrations (1:1)
and targeting non-overlapping epitopes (in different antigenic sites)
were prepared for blind in vivo testing in parallel with HRIG as a
positive control. For this purpose, the volume (ml) delivering
1000 IU for each MoMAb was determined, and mixed with buffer
to the desired final concentration. Two sets of 1:1 MoMAb
cocktails were theoretically adjusted to a total of either 2000 IU
per 5 ml or 2000 IU per 10 ml equalling 400 and 200 IU per ml,
respectively. The latter was also simultaneously blind tested in vitro
as described above but with an incubation period of at least 48 h
to improve robustness of the data.
In vivo testing
In vivo testing was undertaken as a ’’down selection’’ as described
[11,38]. Briefly, 10 female Syrian hamsters in each group were
inoculated with 0.05 ml of RABV virus (Mexican, Thai, or Indian
canine RABV variant) intramuscularly (i.m.) in the gastrocnemius
muscle. Six or 24 hours later, animals were given biologics or PBS
(negative control). Undiluted commercial HRIG (Sanofi-Aventis,
150 IU/ml) or candidate MoMAbs were administered i.m. in the
gastrocnemius muscles in volumes of 50 ml. All i.m. injections were
undertaken using a tuberculin syringe and needle not exceeding 23
gauge. After challenge, animals were observed twice daily and
euthanized at the first clinical signs of rabies (eg. paresis, paralysis,
aggression). Brain tissue was harvested to confirm the rabies
infection using the direct fluorescent antibody (DFA) test [39]. All
animals surviving up to 30 days post infection were euthanized and
tested for rabies as described above. Animal-handling and
experimental procedures were undertaken in compliance with
the CDC’s Institutional Animal Care and Use Committee
(IACUC) guidelines. Ethical approval was obtained for each study
before experiments were initiated (IACUC CDC, USA).
Results
Selection and further characterization of MoMAbs
Five candidate anti-G MoMAbs, E559.9.14 from FLI, Ger-
many, 1112-1 from the Wistar Institute, USA, 62-71-3 from the
CDC, USA, M727-5-1 and M777-16-3 from CFIA, Canada met
the selection criteria and were short-listed to be included in the
cocktail and subjected to further investigation. All hybridomas
were derived from B cells of BALB/c mice immunized
intraperitoneally (i.p.) with either purified G or whole virus
antigen of the ERA vaccine strain of RABV. The hybridomas
were generated with three different fusion partners and at least
three cloning steps (Table 1). Based on information of the cell
culture history for each MoMAb, only approved fetal calf serum
originating from countries being free of foot and mouth disease
(FMD) and TSE, was used in the MAb production.
Candidate MoMAbs were shown to represent two different
subtypes of immunoglobulin, i.e. IgG 1 and IgG 2, as determined
by ELISA or FCA. Sequence analysis of the G gene of the SAD
B19-, CVS-11- and ERA-derived escape mutants of MoMAbs
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E559.9.14, 1112-1, and M777-16-3 showed one or as many as two
amino acid substitutions in the virus G compared to the original
wildtype viruses (Table 1). In vitro cross-neutralisation assays
performed with the escape mutant of MoMAb E559.9.14 showed
that all but one MoMAb recognized epitopes at antigenic site II on
the RABV G. MoMAb 62-7-13 was the only antibody showing a
reaction pattern similar to MoMAb D1, which is known to
recognize conformational epitopes of antigenic site III on G
trimers (Table 2).
The production stability of candidate MoMAbs was determined
after 30 cell passages in serum-containing medium. Only slight
instabilities were observed under laboratory conditions with a few
candidate MoMAbs, but the loss of antibody secretion was less
than 10%, as required if at all (Table 1). Adaptation to serum-free
conditions resulted in a considerable decrease of MoMAb
production. For MuMAb E559.9.14., for example, VNA titres
dropped to 16, 32, and 64 IU/ml when harvested at day 3, 6 and
10, respectively, however, the production remained stable at a
lower level for up to 30 cell passages. Under serum-free medium
conditions, MoMAb M727-5-1 and MoMAb M777-16-3 VNA
titres fluctuated from 22.63, 22.63, 45.25, 32.0 and 32.0 IU/ml to
32.0, 16.0, 45.25, 11.31 and 22.63 IU/ml after 5, 11, 15, 20 and
25 cell passages, respectively. Further characterization of individ-
ual candidate MoMAbs is summarized in Table 1. MoMAb 1112-
1 had to be excluded from further consideration because of
proprietary issues but was kept in the study for comparison.
In vitro studies
A broad in vitro cross-reactivity of the candidate MoMAbs with
RABV and several of the other lyssaviruses was demonstrated at
different virus doses. None of the MoMAbs completely neutralized
the full spectrum of lyssaviruses tested. All five candidate MoMAbs
as well as standard RIG (SRIG) did not neutralize MOKV (gt 3)
and WCBV (putative gt). Also, MoMAb 62-71-3 failed to
recognize DUVV (gt 4), EBLV-1 (gt 5), and MoMAb 1112-1
did not recognize1 lyssavirus (KUHV–putative gt) (Table 3). None
of them recognized LBV. As regards RABVs (gt1), the number of
gt1 RABVs the candidate MoMAbs failed to neutralize ranged
between 2 and 7 (of the 20 RABVs tested). All but one (MoMAb
62-7-13) failed to recognize the Kelev strain of RABV and a skunk
RABV variant originating from California, USA (Table 3).
MoMAbs M727-5-1 and M777-16 required minimally higher
concentrations for neutralization compared to the other candidate
MoMAbs. Interestingly, a cocktail comprising all five candidate
MoMAbs was able to neutralize all viruses tested except MOKV
and WCBV. Identical results were obtained with the polyclonal
SRIG using the same concentrations (data not shown).
Batch production and testing of candidate MoMAbs
under GLP conditions
The minimum yields for purified MoMAbs obtained under GLP
conditions in small-scale cultures were 15 (1112-1, M725-1), 20
(E559.9.14), 25 (62-7-13) and 40 (M777-16-3) mg/L. All purified
MoMAbs produced under these conditions showed two major
bands, at 47 and 20 to 25 kDa on SDS-PAGE corresponding to
isolated heavy and light immunoglobulin chains (Figure 1). The
geometric mean VNA titres of purified candidate MoMAbs (1 mg/
ml) varied from 474 to 10,257 IU/ml. Based on these data, the
immunoglobulin titres (total yield of supernatants) for the five
MoMAb hybridomas was estimated to range between 9,480 and
153,855 IU/L (Table 4).
In vivo and in vitro studies of 1:1 MoMAb mini-cocktail
formulations
The capacity of the 4 MoMAbs to recognize antigenic site II and
the 1 MoMAb to recognize antigenic site III (Table 1) dictated the
preparation of unique 1:1 MoMAb cocktail combinations targeting
non-overlapping epitopes. In particular, MoMAb 62-7-13 (antigen-
ic site III) was combined with each of the four remaining MoMAbs
(antigenic site II). MoMAb cocktail combinations 62-7-13/62-7-13,
62-7-13/1112-1 and MoMAb 62-7-13 (single) were used for
comparison. The results from in vivo MoMAb cocktail combinations
varied slightly (66–100%) but in all cases resulted in protection of
hamsters inoculated with canine RABV variants that was
comparable to HRIG, independent of the concentration (400 IU/
ml or 200 IU/ml) used (Table 5). In comparison to HRIG, the
MoMAb cocktail combinations, adjusted to 2000 IU/10 ml,
neutralized all but two lyssaviruses and the putative lyssavirus gts
in in vitro studies (Table 6). Lyssaviruses not recognized by the mini-
cocktail formulations were LBV and DUVV. In addition, MoMAb
combination 62-7-13/62-7-13 was not able to neutralise EBLV-1.
Discussion
Appropriate mixtures of RABV-specific MAbs generated in vitro
would be a superior alternative to currently employed HRIG and
ERIG for human PEP in rabies endemic areas [16]. Despite the
fact that human hybridomas have been developed [40], the
number of fully characterized HuMAb cocktails suitable for rabies
PEP is still limited [27–29]. Mouse MAbs offer the next best
alternative to HRIG and ERIG since they are able to completely
neutralize RABV and their specific neutralizing activity (IUs per
mg protein) is as much as 2,000 times higher than that of
commercial HRIG [25,36]. Here, we report for the first time, the
identification of three novel combinations of MoMAbs that have a
similar efficacy to HRIG and hence, could form the basis for an
alternative to HRIG or ERIG.
Suitable candidate MoMAbs that form the basis of the
individual cocktails were selected on the basis of stringent criteria,
such as biological activity, neutralizing potency, binding specific-
ity, spectrum of neutralization of natural lyssaviruses, and history
of hybridomas, as applied for a HuMAb cocktail described
recently [27]. These are the requirements for the development of
safe and efficacious MAb alternatives to currently used polyclonal
serum products [41]. The histories of the selected mouse
hybridomas are well documented (Table 1). Alternative biologicals
for PEP including MoMAbs have to overcome a number of
problems associated with the hybridomas, including stability and
Table 2. Neutralization pattern of candidate MoMAbs
(E559.9.14, 62-7-13, 727-5, 777-16) or other MoMAbs (1112-1,
D1) with RABV (E559.9.14 antigenic site II escape mutant,
104FFU/ml) under varying VNA titres (IU/ml).
Titre, IU/ml
MAb 105210.5 0.250.1250.063 0.03
E559.9.14
+++++++++
+
+
+
+
62-7-13
22222222
727-5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
777-16
1112-1
D1
222222222
Absence (2) and presence (+) of viable virus is indicated.
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contamination with potential pathogens [22]. From the informa-
tion on the cell culture history for each MoMAb, the relative risk
of contamination of the hybridomas with FMDV and TSE or and
other adventitious agents was considered minimal because, in all
cases, only approved fetal calf serum originating from countries
free of FMD and TSE was used. Also, concerns associated with
HuMAbs arising from the possibility of a potential spread of
known and unknown human pathogens from hybridoma cells can
be ignored with mouse hybridoma cells. One approach to
overcome the problem of possible pathogen contamination of
the MAb preparations, while maintaining the high binding
affinities produced by somatic mutation of the B cell in response
to antigenic stimulation, however, is to clone and express the
immunoglobulin genes from the monoclonal hybridoma cell line
in heterologous systems [22,42,43].
The production stability observed after 30 cell passages using
serum-containing medium was satisfactory and met the require-
ments, as none of the mouse hybridomas of the candidate
MoMAbs showed a considerable loss of VNA secretion. However,
the optimal supernatant harvest time, taking into account cell
viability, can still be optimised. Production of candidate MoMAbs
under GLP conditions, as undertaken by a WHO service
manufacturer, showed that the hybridoma yields could be
improved at least 10–20 times in bioreactors (Table 4). However,
the use of serum-free medium resulted in a decrease in the VNA
production. Hence, adaptation to an appropriate commercially
available serum-free medium should be the subject of further
investigation. Based on experience, serum-containing medium
might be preferential for conservation of the hybridomas, as this
will result in a better and more stable survival rate of the
Table 3. In vitro neutralization pattern of individual candidate MAbs.
Lyssaviruses gtVirus dose (log10)SRIG M777-16 M727-562-7-13 (03-043)62-7-13 (03-041)62-7-13 (03-026) 1112-1E559
Bobcat, USA140.125 1.05.0 0.063n.d.n.d. 0.25
+
CVS-11140.063 2.05.00.125n.d.n.d.0.250.25
Dog, Azerbaizhan140.25
++
0.25n.d.n.d. 1.010.0
Dog, Ethiopia14 0.1255.05.00.5n.d.n.d. 0.50.5
Dog, India14 1.05.0
+
+
0.125n.d. n.d.1.00.25
Dog, Mexico14 0.510.0 0.5n.d.n.d. 2.00.063
Dog, Nepal140.25 2.010.00.125 n.d.n.d. 0.125 0.125
Dog, Turkey14 0.125
++
0.125n.d.n.d.
+
0.5
Fox, Eastern Europe 14 0.51.05.00.125n.d.n.d.0.250.125
Fox, Europe140.125 1.05.05.0n.d. n.d. 0.50.25
Kelev, Israel14 0.25
++
0.063 n.d.n.d.
++
Polar fox, Norway140.510.0 10.0 0.5n.d.n.d. 0.5 0.25
PV14 0.252.0 10.0
+
n.d. n.d.0.5 0.063
SAD B1914 0.25 5.010.02.0n.d. n.d.0.5 0.25
Wolf, Bosnia14 0.125 2.0
+
0.125n.d.n.d.0.1250.25
EBLV-1, Germany541.0 1.010.0
+
n.d.n.d.0.250.5
EBLV-2, UK642.05.010.00.125n.d.n.d.0.50.25
Arctic fox, AK, USA12 0.016 0.014 0.016 0.0190.1140.0120.01360.016
CVS-11120.0160.014 0.016 0.019 0.1140.0120.0136 0.016
CVS-11120.0160.014 0.0160.0190.002 0.0020.0136 0.016
Gray fox, TX, USA120.0160.0140.0160.0030.1140.0020.0680.016
Raccoon, SC, USA120.08 0.0140.080.0030.0572 0.012
+
+
+
+
0.08
Skunk, CA, USA12 0.08
++
0.003 0.002 0.002
+
Skunk, SC, USA12 0.0160.0140.016
+
+
+
+
+
+
+
+
+
+
+
+
0.016
MOKV, Africa32
++++
DUVV, Africa 420.08 0.014 0.0160.0680.016
EBLV-1, Europe520.080.0140.0160.01360.016
EBLV-2, Europe620.080.0140.0160.0030.0020.0020.01360.016
ABLV, Australia720.0160.0140.0160.0030.1140.0120.0680.016
ARAV, Asia 2 0.080.0140.080.019 0.0020.0020.340.016
IRKV, Asia20.080.0140.0160.0190.1140.0020.0680.016
KHUV, Asia20.080.0140.0160.0030.0020.002
+
+
0.016
WCBV, Europe2
+++++++
Tests were conducted in comparison to standard rabies immunoglobulin (SRIG) against lyssa- (gt 1–7) and putative lyssavirus gts. MoMAbs M777-16 and M727-5 were
used purified and the remaining as cell culture supernatants. For MAb 62-7-13, three different harvests were tested. Figures in boxes show the minimum MoMAb
concentration in IU/ml at which complete neutralization was observed. Boxes with cross (+) represent presence of viable virus.
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hybridoma cells due to the protective function of the fetal calf
serum. Isotyping showed the candidate MoMAbs to be of the IgG
isotype (subtypes 1 & 2) and thus, ideal with respect to PEP, since
IgGs are expected to have a longer half-life in vivo than other
immunoglobulin types [44].
Our in vitro efficacy studies of candidate MoMAb demonstrated
their capacity to neutralize a broad spectrum of RABV and other
lyssaviruses of phylogroup I (Table 3), consistent with previous
studies demonstrating cross-neutralization and cross-protection
[1,45]. The lack of cross-neutralization with phylogroup II viruses
and WCBV was also expected from the phylogenetic distance,
which correlates with previous studies [1,38]. However, this
limitation is less important as human infections with these
genotypes are extremely rare and do not represent a major threat
for public health. Previous studies comparing the antigenic
phenotype of diverse RABV isolates showed that different
neutralizing epitopes were shared between Pitman Moore (PM)
and other RABV strains and supports our observations [46,47].
However, none of the candidate MoMAbs alone was able to
neutralize all of the RABVs tested. One explanation might be the
different virus dose used in the in-vitro neutralisation assays, since
higher concentrations of MoMAbs were needed to neutralize the
higher virus dose (Table 3 and 6). On the other hand, RABV
strains that were not neutralized in vitro may represent natural
escape mutants if individual candidate MoMAbs were unable to
recognize specific epitopes on the G. In contrast, a cocktail
comprising all candidate MoMAbs conferred protection in the
same model (data not shown). These data emphasise the need for
an ideal therapeutic modality to consist of a mixture of at least two
MoMAbs to ensure that all known RABV strains are targeted with
a standardized reagent [16,22,48].
In addition to the broad spectrum of virus neutralization that
these MAbs, in general, are capable of, candidate MoMAbs should
target distinct, non-overlapping epitopes and should not compete
for binding to the RABV G. Of the selected MoMAbs, all but one
recognized antigenic site II on the RABV G, as shown by
sequencing the epitope binding sites at the G-gene level and the
generation of MoMAb-specific escape mutants in combination
with in vitro cross-neutralization assays (Table 2). This indicated
that MoMAb 62-7-13 (CDC, USA) was an essential component
for a unique standardized MoMAb cocktail combination. Since
one candidate MoMAb had to be excluded from further
consideration because of intellectual property ownership, this
resulted in three novel combinations of MoMAbs cocktails
targeting non-overlapping epitopes present in antigenic sites II
and III (Table 6).
Further characterization of the functional properties of the three
unique MoMAbs cocktail combinations and their capacity to
prevent the spread of RABV both in vitro and in animal models will
be assessed in future studies. As these MoMAbs cocktail
combinations can be considered as an alternative to HRIG and
ERIG for PEP treatment in developing countries, the principal
focus of the in vitro and in vivo studies remains on neutralization of
RABVs isolated from dogs from different geographical areas ([4],
Tables 3 and 6). In the blind in vitro efficacy studies of the three
MoMAb cocktails, a range of doses was determined in an effort to
Figure 1. Coomassie blue staining of purified MoMabs (5 mg/
well) demonstrating appropriate size of light chain and heavy
chain. Ma–molecular weight marker in kD; lane1, E559 (batch # 603-02);
lane 2, 62-7-13 (batch # 604-26); lane 3, 1112-1 (batch # 604-26); lane 4,
M777-16-3 (batch # 605-03); lane 5, M777-16-3 (batch # 605-12); lane 6,
M727-5-1 (batch # 605-03); lane 7, M727-5-1 (batch # 605-19).
doi:10.1371/journal.pntd.0000542.g001
Table 4. Neutralization results obtained after batch production under GLP conditions.
Purified MoMAbsSupernatant
MoMAbsAntigen content (g/ml)GEO VNA (IU/ml)SDMin MaxVNA (IU/mg)VNA (IU/mg)
E559.9.41474.29 86.39375.04532.58474
.9,480
1112-11 5990.38222.135860.266246.875990
.89,850
62-7-131501.28251.30337.38790.61501
.12,525
M727-5-1110256.792088.87 8482.0012558.7510257
.153,855
M777-16-31 1962.40511.871534.12 2529.311962
.78,480
Controls
WHO SRIG0.500.000.500.50
Negative control0.080.00 0.07 0.08
Geometric mean (GEO) VNA titres and standard deviation (SD) of 1 mg/ml of purified MoMAbs as determined by RFFIT in three independent tests and subsequent
estimation of the yield of supernatant of the five hybridomas in comparison to a negative control and the WHO standard rabies immunoglobulin (SRIG).
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Table 5. Protection of hamsters challenged with RABV following treatment with MoMAb combination cocktails during three
independent in vivo studies.
MAb/MAb cocktailVolume delivering 1000 IU/mL400 IU/ml200 IU/ml
Mex2004Protection in %Thai2006Protection in %India2008Protection in %
PBSn.d. n.d.5/9
62-7-13/E5590.34/0.458/988 6/966 7/9 77
62-7-13/M7770.34/0.06 9/9 1009/9100 8/9 88
62-7-13/M7270.34/0.018/9 886/9668/9 88
62-7-13/62-7-13 0.34/0.34 9/91005/9 559/9 100
Controls
62-7-13/1112-10.34/0.089/9100 7/977n.d.
62-7-13 only 0.349/91001/911 n.d.
HRIG positive control4/9445/9 557/9 77
HRIG negative control 3/90/9 3/9
Survivorship of hamsters after challenge with a Mexican (2004), Thai (2006), or Indian (2008) canine RABV variant and subsequent treatment with 1:1 cocktail
formulations of MoMAbs to simulate passive immunization in PEP.
doi:10.1371/journal.pntd.0000542.t005
Table 6. In vitro neutralization pattern of equal amounts of MoMAbs in combination cocktails.
Virus gt
Virus dose
(log10)
Incubation time
(days)SRIG PBS62-7-13/E55962-7-13/M77762-7-13/M727 62-7-13/62-71-3
Bobcat, USA1420.125
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.1250.125 0.1250.125
Dog, Azerbaizhan142 0.250.250.250.250.25
Dog, Ethiopia142 0.1250.25 0.50.252.00
Dog, India1421.000.1250.125 0.50.5
Dog, Mexico142 0.50.250.25 0.25 0.5
Dog, Nepal1420.25 0.25 0.250.250.5
Dog, Turkey142 0.125 0.125 0.250.250.25
Fox, Eastern Europe1420.50.125 0.125 0.1250.5
Fox, Europe1420.125 0.250.1250.252.00
Polar fox, Norway142 0.5 0.1250.125 0.1250.25
Wolf, Bosnia1420.1251.00 1.001.00 1.00
EBLV-1, Germany542 1.000.5 0.250.5
EBLV-2, UK6422.000.50.51.001.00
Arctic, Canada127n.d. 0.009 0.0050.009 0.018
Big Brown Bat, Canada127n.d.0.0020.0050.003 0.026
CVS-11127 n.d.0.0130.0050.004 0.005
Dog, Sri Lanka127n.d.0.007 0.014 0.0090.026
ERA127n.d.0.0130.0190.0120.204
Mongoose, Africa127n.d.0.0090.0140.0090.026
Silver Haired Bat, Canada 127n.d. 0.0070.007 0.0060.013
Vampire Bat, Latin America127n.d.0.0130.0540.035 0.051
LBV, Africa227n.d.
+
+
+
+
+
+
+
+
+
DUVV, Africa 427n.d.
EBLV-1, Europe527n.d.0.0190.0140.035
EBLV-2, Europe627n.d.0.0030.0270.0090.036
ABLV, Australia727n.d.0.0070.0030.0040.005
In vitro neutralization pattern of equal mixes of MoMAbs in combination cocktails adjusted to 2000 IU/10 ml in comparison to SRIG against lyssaviruses of gt 1–7 and
putative lyssavirus gts. Figures in boxes show the minimum MoMAb concentration in IU/ml at which complete neutralization was observed. Boxes with cross (+)
represent the presence of viable virus.
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determine the appropriate doses that could be used in animal
studies. In animals, antibody administration may range from 2–
4 IU per animal for small-bodied species, 200–400 IU per
medium-size animals and up to 2000–4000 IU per large animal
(or human). The actual formulations may be ,150–200 IU/ml or
more. However, in vivo, these rarely reach .1 IU/ml of serum.
Thus, the range was considered optimal from ,0.6 to 2.5 IU/ml.
The MoMAb cocktail combinations showed neutralisation within
the suggested IU range (Table 6). In vivo, all three MoMAb cocktail
combinations resulted in protection rates (66–100%) for hamsters
challenged with canine RABV variants that were comparable to
HRIG (Table 5). Similar observations were made recently with
other single MoMAbs depending on the strain of the virus [25]. As
in this study, the in vivo testing was undertaken for ’proof-of-
concept’. Clearly, further studies should be undertaken to provide
additional statistical evidence.
Our preliminary in vivo studies with MoMAbs cocktails provided
encouraging results. However, it could be presumed that the use of
such antibodies in humans might have limitations, as with ERIG,
because of the potential of foreign proteins to cause side effects.
Even antibody fragments, which are less likely to be recognized as
foreign could present problems as they seem less stable in vivo than
whole antibodies [17]. Human MAbs for rabies PEP would be
preferential; however, MoMAbs can be readily humanized [49].
Also, their unknown compartmentalization, half-life as well as
immunogenicity in humans, is supposed to prevent MoMAbs from
being ideal replacements for the existing reagents [16]. Despite
these limitations, the end-product of the WHO project would be of
an improved quality over ERIG.
The approach used here to develop and establish a suitable
MoMAb cocktail combination of a minimum of two anti-G
MoMAbs able to replace RIG for human PEP against rabies was
based on the ultimate goal to make a product, which can be used
in developing countries. Although the WHO project should be
seen within the context of wider biological product market
competitiveness, the ’uniqueness’ of the WHO project as described
here is in the preferential conditions under which the product
would have to be produced and made available to the public sector
of rabies-endemic countries, particularly of the developing world
(e.g. production costs). Therefore, the three novel MoMAb
combination cocktails can be considered a less expensive
alternative for prophylactic use to prevent rabies in humans.
Currently, both phase I safety trials of the MoMAb product and
humanization of the MoMAbs are under consideration.
Acknowledgments
We thank staff at the CDC for their scientific input and technical expertise
to date, especially Richard Franka, Lauren Greenberg, Felix Jackson,
Boonlert Lumlertdacha, and Pamela Yager. The authors would like to
thank William Wunner for his valuable suggestions for improvements and
the final editing of the manscript.
Author Contributions
Conceived and designed the experiments: TM BD HCE ARF FXM CER
NT AIW MPK. Performed the experiments: TM ARF CFG JK CER
AIW. Analyzed the data: TM CF CFG CER AIW. Contributed reagents/
materials/analysis tools: TM BD HCE CFG CER NT AIW. Wrote the
paper: TM HCE ARF CF.
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