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Virus Research
journal homepage: www.elsevier.com/locate/virusres

Review

Antibody-mediated neutralization of African swine fever virus: Myths and facts
José M. Escribano ∗ , Inmaculada Galindo, Covadonga Alonso
Departamento de Biotecnología, INIA, Autovia A6 Km 7, 28040 Madrid, Spaina r t i c l e

i n f oArticle history:
Available online xxx
Keywords:
African swine fever virus
Antibody
Neutralization
Vaccinea b s t r a c t
Almost all viruses can be neutralized by antibodies. However, there is some controversy about antibodymediated neutralization of African swine fever virus (ASFV) with sera from convalescent pigs and about
the protective relevance of antibodies in experimentally vaccinated pigs. At present, there is no vaccine
available for this highly lethal and economically relevant virus and all classical attempts to generate a
vaccine have been unsuccessful. This failure has been attributed, in part, to what many authors describe
as the absence of neutralizing antibodies. The ﬁndings of some studies clearly contradict the paradigmof the impossibility to neutralize ASFV by means of monoclonal or polyclonal antibodies. This review
discusses scientiﬁc evidence of these types of antibodies in convalescent and experimentally immunizedanimals, the nature of their speciﬁcity, the neutralization-mediated mechanisms demonstrated, and thepotential relevance of antibodies in protection.
© 2012 Elsevier B.V. All rights reserved.

Contents
1.
2.
3.
4.
5.
6.
7.
8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Antibodies are relevant in the ASFV-induced protective immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vitro neutralization mechanisms of ASFV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors inﬂuencing the susceptibility of ASFV to neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The persistent non-neutralized virus fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Proteins mediating the induction of ASFV-neutralizing antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Antibodies directed to the dynein-binding domain of protein p54 neutralize ASFV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
African swine fever (ASF) is a viral disease that affects populations of wild boars and domestic pigs. In the latter it causes
hemorrhagic fever, leading to high mortality rates and consequently signiﬁcant economic losses. ASF is brought about by a large
DNA virus, African swine fever virus (ASFV), which is to date thesole member of the family Asfarviridae (Dixon et al., 2011). However, the discovery of novel viral sequences in human serum andsewage has recently been reported. These sequences are clearly
related to the asfarvirus family but are highly divergent from ASFV.
The detection of these sequences suggests that asfarviruses show
greater genetic diversity than previously thought and raises thepossibility of human infection by these viruses (Loh et al., 2009).

∗ Corresponding author. Tel.: +34 913473917; fax: +34 913478711.
E-mail address: escriban@inia.es (J.M. Escribano).

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There is no vaccine currently available to prevent ASF. Attempts toimmunize animals using vaccine formulations prepared by conventional means and comprising infected cell extracts, supernatants ofinfected pig peripheral blood leukocytes, puriﬁed and inactivatedvirions, infected glutaraldehyde-ﬁxed macrophages, or detergenttreated infected alveolar macrophages failed to induce protective
immunity (Coggins, 1974; Forman et al., 1982; Mebus, 1988).
A number of recent reviews have addressed the role of cytotoxic
T cells in immune defense against infectious agents; however, little attention has been devoted to an analogous role for humoral
immunity, the component of the immune system that meets the
microorganism at the portal of entry and interacts with it in all
the body compartments that it subsequently invades. Regardlessof the mechanism involved in virus inactivation, the attachmentof antibodies to virus particles lead to loss of infectivity. However,
the existence of non-neutralizing antibody, which binds to virus
without diminishing infectivity, has long been recognized. Thereis a lack of basic knowledge about neutralization mechanisms in

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many animal viruses and about the relationship between in vitro
neutralization and natural protection. A common misperception isthat antibodies act solely by preventing the attachment of neutralized virus to target cells (Dimmock, 1993, 1995; Li et al., 1994).
Antibodies, however, exert their neutralizing action by a variety
of mechanisms, some of which are poorly understood. Neutralizing antibodies vary enormously in the rate and efﬁciency withwhich they act. The observation that neutralization rate constants
can vary >1000-fold not only between distinct viruses but also
between different antibodies to the same antigenic sites on thesame virus suggests that the categorical capacity of an antibody
or antiserum to neutralize is an over-simplistic correlate of protection (Krause et al., 1997). Moreover, individual virions may have
many neutralizing epitopes, and in natural infections each of these
may be available to bind to one of the variety of antibody speciﬁcities present at various concentrations, depending on the individual.
Neutralization may be synergistic in such situations; however, it
might also be antagonistic (i.e., the binding of antibody to one neutralizing epitope might interfere with the binding of antibody toanother one) (Krause et al., 1997). In addition, some antibodies
appear to up-regulate cell infection in vitro, a phenomenon referred
to as antibody-dependent infection enhancement. Enhancement
has been detected in many viral systems, usually with antibodies toenvelope epitopes and typically with neutralizing antibodies (i.e.,
those that normally neutralize at adequate concentration) whenbound at sub-neutralizing concentrations (Morens, 1994).
At present, it is widely accepted that nearly all viruses haveneutralization sites. In the past, it was considered that only ASFV
and Marburg and Ebola viruses lacked the ability to be neutralizedby virus-speciﬁc antibodies (Dimmock, 1993). The last decade hasrevealed that ﬁloviruses (Marburg and Ebola viruses) are efﬁciently
neutralized by monoclonal and polyclonal antibodies (Hevey et al.,
2003; Maruyama et al., 1999; Shedlock et al., 2010) and that these
antibodies have protective properties (Hevey et al., 2003; Takada
et al., 2007; Warﬁeld et al., 2004). Early experiments with ASFV
were structured and interpreted on the basis of the hypothesis thatneutralizing antibodies are not induced by this virus (Hess, 1981;
Viñuela, 1985).
Since the 80s, several strategies using viruses attenuated by passage in tissue culture (Gómez-Puertas et al., 1998; Ruiz Gonzalvo
et al., 1986a,b) or using natural low virulence isolates (Boinas et al.,
2004; Denyer et al., 2006; King et al., 2011; Leitao et al., 2001) havebeen developed to obtain pigs that are resistant to ASFV. These
strategies allow study of the immune mechanisms responsible forprotection. In ASF, several studies on the implication of antibodies
in protection have convincingly demonstrated that they may delay
onset of the disease and reduce viremia titers, two aspects that, toa great extent, determine the survival of pigs after infection. All ofthese issues will be discussed in this review.
2. Antibodies are relevant in the ASFV-induced protective
immune response
Generally speaking, both cellular and humoral immune
responses contribute to protection against viral infections and tothe clearance of viruses from infected individuals. Speciﬁc experiments are required to discern the contribution of the mechanisms
implicated in protection. Experimental vaccination protocols to
obtain protected animals are the ﬁrst step to analyze immune
mechanisms, giving key information on which to develop in future
strategies aimed at obtaining a licensed vaccine. In the case of ASF,
only a few experimental models have been established (GómezPuertas et al., 1996; King et al., 2011; Leitao et al., 2001; Lewis
et al., 2000; Oura et al., 2005; Ruiz Gonzalvo et al., 1986b; Stoneet al., 1968; Zsak et al., 1993). Attenuated virus isolates OUR/T88/3
and E75CV1 -4 have been used to study cellular and humoral

protective immune responses. Pigs exposed to OUR/T88/3 andthen depleted of CD8+ lymphocytes were no longer fully protectedfrom virus-related OUR/T88/1 challenge. This observation indicated that CD8+ lymphocytes play a critical role in the protective
immune response to ASFV infection and that anti-ASFV antibody
alone, from OUR/T88/3 infection, was not sufﬁcient to protect pigs
from OUR/T88/1 challenge. In contrast, antibodies obtained frompigs that had recovered from infection with one of the viruses
most used in experimental protection experiments, denominated
E75CV1 -4 virus, a Spanish strain (E75) adapted to grow in CV1 cells
and propagated in pig macrophages (Barderas et al., 2001; GómezPuertas et al., 1996, 1998; Onisk et al., 1994; Ruiz Gonzalvo et al.,1986a,b) appeared to confer protection against ASFV infection.
Sera from convalescent swine infected with this attenuated virus
isolate neutralized the infectivity of virulent ASFV isolates E75,
E70, Lisbon 60, Malawi Lil 20/1 and a low-passage tissue cultureadapted variant of E75, namely E75CV/V3, by 86–97% in Vero andmacrophage cell cultures (Zsak et al., 1993). The role of anti-viral
antibodies in homologous protective immunity to a virulent ASFV
strain, E75, was examined by passive transfer experiments inswine. Eighty-ﬁve percent of the animals that received anti-ASFV
immunoglobulin survived challenge infection. In contrast, 100%
mortality was observed in the experimental group that received
control immunoglobulin sera fractions or phosphate-bufferedsaline. With the exception of a signiﬁcantly delayed and transient
fever response, the animals who received anti-ASFV antibodies
remained clinically normal following challenge, whereas thecontrol group presented clinical ASF on day 4 post-challenge. In
addition, a signiﬁcant 3-day delay in the onset of viremia and a
10,000-fold reduction in both mean and maximum virus titers wereobserved for animals given anti-ASFV immunoglobulins. These
results indicate that anti-ASFV antibodies alone protect swine fromlethal infection with virulent ASFV (Onisk et al., 1994). Furthermore, they support the view that the antibody-mediated protective
effect is an early event that effectively delays disease onset (Onisk
et al., 1994). Other reports concerning the role of antibodies in ASFV
protection showed that when these antibodies are transferredthrough colostrum, they also confer to suckling piglets a degree ofprotection against viral challenge (Schlafer et al., 1984a,b). Fig. 1
shows a schematic representation of in vivo antibody-mediatedprotection against ASFV described in the literature.
3. In vitro neutralization mechanisms of ASFV
Little work has been carried out on ASFV neutralization, presumably because early experiments were structured and interpreted
on the basis of the hypothesis that neutralizing antibodies are
not induced by this virus (Hess, 1981; Viñuela, 1985). However,other authors have demonstrated that a number of isolates of
ASFV are neutralized by immune sera from convalescent swine andmonoclonal antibodies (Borca et al., 1994; Gómez-Puertas et al.,
1996; Ruiz Gonzalvo et al., 1986a,b; Zsak et al., 1993). Those studies that reported neutralization described a persistent fraction of
non-neutralized virus of about 10%. This fraction was not demonstrable by an infection inhibition test, in which swine immune sera
inhibited infection by the homologous and sometimes heterologous viruses in pig macrophages (Ruiz Gonzalvo et al., 1986b).
The use of conventional plaque reduction assays to measure antibody neutralization of ASFV presents frequent difﬁculties
because many strains, especially low-passage viruses, show anabsence or delay in plaque formation. Even more problems
are encountered when performing neutralization assays in pig
macrophages, the natural host cells. This would explain why, inneutralization assays, most authors use viruses that are highly
adapted to cell lines. This point will be discussed further below
because it has important consequences for the interpretation ofPlease cite this article in press as: Escribano, J.M., et al., Antibody-mediated neutralization of African swine fever virus: Myths and facts. Virus
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Fig. 1. Schematic representation of in vivo protection experiments described in the literature and involving antibodies taken from pigs that had recovered from ASF.

results. To overcome this problem, tests based on genetically
modiﬁed viruses expressing chromogenic marker genes, such as ␤glucuronidase or ␤-galactosidase, were developed (Gómez-Puertas
et al., 1995) to facilitate in-depth studies of the antibody neutralization mechanisms of ASFV. Neutralization assays could now be
performed with these viruses, either low- or non-passaged in cell
culture, and experiments took less than a third of the time neededusing non-recombinant viruses. In addition, small plaques could
be detected more accurately by color contrast. These recombinantviruses also permitted differentiation by chromogenic staining ofindividual infected pig macrophages, thus facilitating the comparison of neutralization assays results in these primary cultures andcell lines.
The abovementioned recombinant viruses expressing marker
genes have been used in studies that address the presence ofneutralizing antibodies in convalescent pigs after infection witha range of attenuated viruses (Gómez-Puertas et al., 1996). Heatinactivated sera from these pigs reduced the infectivity of several
low cell culture-passage viruses between 87 and 100%. As previously mentioned, the persistent fraction of non-neutralized virus(4–13%) has been described (Ruiz Gonzalvo et al., 1986a; Zsak et al.,1993) and has also been found in other enveloped viruses (Asheand Notkins, 1967; Jackson et al., 1991; Poumbourios et al., 1990).
Neutralizing antibody induction seems to be an early event during infection with attenuated viruses as sera from convalescentpigs, in many cases, neutralized more than 50% of virus infectivity at day 9 post-infection. Neutralizing antibodies in vaccinatedpigs reached a plateau at the end of the second week after infection(Gómez-Puertas et al., 1996).
Antibodies from pigs vaccinated with attenuated viruses use atleast two mechanisms to neutralize virus infectivity in cell lines andmacrophage primary cultures. Virus binding experiments to Verocells or pig alveolar macrophages is a saturable mechanism (Alcami
et al., 1989) and reached a plateau 120–240 min after the reaction was started (Gómez-Puertas et al., 1996). Antibody-mediatedblocking of virus attachment to cells has been demonstrated experimentally by adding the antibodies before or after virus attachment
to cells at 4 ◦ C, a temperature at which virus internalization is
inhibited (Fig. 2A). A relationship between inhibition of virus binding to susceptible cells and virus neutralization has been reported.

Radiolabeled virus binding in the presence of anti-ASFV sera wasinhibited by more than 80% at the time of maximum binding in
the absence of neutralizing antibodies, both in Vero cells and in
macrophages (Gómez-Puertas et al., 1996). Parallel neutralization
experiments demonstrated that these sera reduce radiolabeledvirus infectivity more than 90%. These results conﬁrmed the existence of an ASFV neutralization mechanism in which antibodies
inhibit the binding of the virus to cells.
A second ASFV neutralization mechanism has also been demonstrated by inhibiting radiolabeled virus internalization in thepresence of immune sera in Vero cells or pig alveolar macrophages
determining the detachment of non-internalized virus by proteinase K treatment (Fig. 2B). While the internalization of the virus
previously attached to the cells at 4 ◦ C was not affected by addition

Fig. 2. Determination of ASFV neutralization mechanisms. (A) Percentage ofinhibition of radiolabeled ASFV attachment to Vero cells after incubation with representative negative control or immune pig sera. (B) Percentage of radiolabeled ASFV
detachment by proteinase K treatment of infected Vero cells with the virus at 4 ◦ C(attached but not internalized) and then incubated with representative negative
control or immune pig sera.

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of a pre-immune control serum, more than 90% of the virus was
released from the cells by protease treatment 4 h after the temperature of the cells was reversed from 4 to 37 ◦ C in the presence ofneutralizing sera (Gómez-Puertas et al., 1996). No signiﬁcant differences were observed between the experiments performed with
Vero cells and those with pig macrophages. These observations
revealed a second ASFV neutralization mechanism that abrogates asecond step of the replication cycle involving virus internalization.
Experiments carried out with ASFV cannot rule out that one ofthese mechanisms may be predominant at different antibody-tovirus ratios, as described for other virus models (Sune et al., 1990).
However, under the conditions described in this virus model, thetwo mechanisms showed almost equivalent efﬁciency. About 80%
of virus binding was inhibited and more than 90% of the virus
was not internalized in the presence of neutralizing antibodies(Gómez-Puertas et al., 1996). The combination of the two mechanisms neutralized more than 95% of virus infectivity. In summary,neutralization of ASFV may result from the inhibition of at least
two steps of its replication cycle, namely virus attachment andinternalization.
4. Factors inﬂuencing the susceptibility of ASFV toneutralization
In the past, it was considered that ASFV could not be neutralized by antibodies (Hess, 1981; Viñuela, 1985). Interestingly, a
study published by Zsak et al. (1993) showed that serum from aconvalescent swine to a particular ASFV isolate (E75) neutralizes
the infectivity of various virulent ASFV isolates (E75, E70, Lisbon 60, Malawi Lil 20/1) and low-passage tissue culture-adaptedvariants by 86–97% in Vero and macrophage cell cultures. Unexpectedly, these immune sera failed to neutralize high-passage
tissue culture-adapted ASFV variants, including Lisbon 60, Haiti,
Dominican Republic I, Dominican Republic II, and Brazil II. A similar result was obtained with a neutralizing monoclonal antibody(135D4), which reacted with all viruses. On the basis of these ﬁndings, the authors proposed that tissue culture adaptation of ASFV
isolates is related to the loss of speciﬁc determinants associatedwith virus neutralization.
ASFV propagation in cell lines has been shown to modify key
genetic and phenotypic properties of the virus, such as replication
capacity in pig macrophages, the natural host cell (Alcaraz et al.,
1992; Rodriguez et al., 1994). However, dramatic changes in
the antigenic conformation of several virus proteins involved inneutralization cannot be expected in all viruses propagated incell lines. A later study (Gómez-Puertas et al., 1997) conﬁrmedthat low- and high-passage viruses differ in their susceptibility
to neutralization (Fig. 3A). Those authors demonstrated thatdifferences in susceptibility were not due to antigenic variabilityof critical epitopes and revealed the relevance of the phospholipid
composition of ASFV virions; this composition was found to be
distinct depending on the number of cell culture passages. A
comparative analysis of phospholipid composition of the membranes of low- and high-passage viruses revealed differences in
the relative amount of phosphatidylinositol in these two groups,
independently of the cells in which the viruses were cultured(Fig. 3B). Further puriﬁcation of low- and high-passage viruses
by Percoll sedimentation showed differences in the phospholipid
composition identical to those found with partially puriﬁed viruses
and conﬁrmed the susceptibility of these viruses to neutralization.
The incorporation of phosphatidylinositol into membranes ofhigh-passage viruses rendered a similar neutralization susceptibility to low-passage viruses, in which phosphatidylinositol is amajor phospholipid (Fig. 3C). In contrast, other phospholipids did
not interfere with high-passage virus neutralization, suggestingthat this membrane component is essential for correct epitopeFig. 3. Relevance of phospholipid composition of ASF virions for susceptibility toantibody neutralization. (A) Neutralization susceptibility of low- and high-passage
ASFV isolates in cell lines, measured by a plaque number reduction assay in Verocells and using sera from convalescent pigs.The ﬁgure shows the media and deviation standards of neutralization data obtained with three different viruses belonging
to each group. (B) Phospholipid composition of viral membranes from representative puriﬁed low- and high-passage ASFV isolates. Differences are determined by the
relative amounts of phosphatidylinositol incorporated. (C) Modiﬁcation of neutralization susceptibility of low- and high-passage representative ASFV isolates afterincorporation (+) or removal (−) of phosphatidylinositol from viral membranes.

presentation to neutralizing antibodies. In addition, the removal ofphosphatidylinositol from a low-passage virus by a speciﬁc lipasetransformed this virus from neutralizable to non-neutralizable(Fig. 3C). These data reveal the importance of the lipid compositionof viral membranes for protein recognition by antibodies and may
account in part for past difﬁculties in reproducibly demonstrating
ASFV-neutralizing antibodies using high-passage viruses.
Given the differential neutralization patterns between highand low-passage ASFVs, great care should be taken when selecting the virus isolate to be used in neutralization studies. Other
examples of neutralization dependence on passage history of theviruses and/or on the host system have been described (Baldinotti
et al., 1994; Grady and Kinch, 1985; Kim et al., 1994). As in otherviral systems (Luan et al., 1995), ASFV directs the phospholipid
composition of its membrane, since the relative percentages ofphospholipids in the membranes of low- and high-passage virusesgrown for several passages in the same cells differ (Gómez-Puertas
et al., 1997). In Aleutian disease virus, the lipid composition of virions conditions their capacity to be neutralized (Stolze and Kaaden,1987). The presence of high amounts of phosphatidylinositol in
low-passage virions suggests that ASFV membranes are derived
from the endoplasmic reticulum (ER) compartment of the hostcell. This notion is supported by the phospholipid composition of
newly formed virions, which is similar to the known membranecomposition of the ER, where essentially all phosphatidylinositol issynthesized (van Meer, 1989). However, in contrast, high-passage,
non-neutralizable virions have a phospholipid composition that isintermediate between that of the ER membrane and that of later
secretory compartments (van Meer, 1989). This ﬁnding suggeststhat the membrane formation mechanisms in low- and highpassage viruses differ (Sodeik et al., 1993).
Phospholipids are crucial for the antigenic properties of hepatitis B surface antigen (HBsAg). All epitopes of this protein deﬁned bya panel of monoclonal antibodies show variation of reactivity afterreconstitution with acidic phospholipids (Gómez-Gutierrez et al.,
1994, 1995). Electrostatic interactions between HBsAg proteins andacidic phospholipids are partly responsible for the complete recovery of antigenic activity. Those authors proposed that the antigenic
activity is dependent on the physical state of the phospholipid
moiety. Once the conformation of the antigen in membranes is

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mutant viruses from the persistent non-neutralized ASFV fraction
were also unsuccessful. Nevertheless, competition experiments
between sera demonstrated that antibodies present in sera showing the persistent fraction abrogates the full neutralization (100%)
mediated by other sera (Fig. 4B). These results strongly suggest thatthe induction of blocking antibodies during ASFV infection could
be one of the main causes of the persistent surviving virus fraction
observed in neutralization assays and could also explain the persistent infections observed in some convalescent pigs. However, itcannot be discarded that other aspects related to the virus infection,such as direct transmission of the virus cell to cell, could inﬂuence
the scape of viruses to the antibody-mediated neutralization.

6. Proteins mediating the induction of ASFV-neutralizing
antibodies

Fig. 4. Demonstration of the presence of neutralization blocking antibodies in serafrom ASF convalescent pigs and the implication of these antibodies in a persistentnon-neutralized virus fraction. (A) Determination of the persistent non-neutralized
fraction of a representative ASFV isolate observed in neutralization assays using serafrom convalescent pigs (1–3). Only serum number 3 neutralized 100% of virus infectivity. (B) Interference of antibodies present in a serum showing a typical persistent
fraction with the complete neutralization of serum number 3 (serum number 1).
The order used in the incubation of sera 1 and 3 with the virus determined the nonneutralized persistent virus fraction found at the end of the neutralization assays.
The previous incubation of the virus with the partially neutralizing serum 1 abrogated the complete neutralization observed with serum number 3. This observationsuggests the presence of blocking antibodies in serum 1.

established, additional interactions by the various phospholipids
may alter the pattern of antigenicity, as shown with HBsAg (GómezGutierrez et al., 1994, 1995) or with epitopes involved in ASFV
neutralization.
5. The persistent non-neutralized virus fraction
A persistent non-neutralized ASFV fraction of about 10% is foundwith most convalescent swine sera in in vitro neutralization assays(Gómez-Puertas et al., 1996; Ruiz Gonzalvo et al., 1986a; Zsak et al.,1993) (Fig. 4A). The inability of this virus fraction to be completely
neutralized by antibodies could lead to a very frequently describedchronic ASFV infection in pigs, in which infectious virus persists
even in the presence of an excess of neutralizing antibodies.
In other viral models, this persistent fraction has been attributed
to several factors, including the following: formation of viral aggregates that are non-accessible to antibodies (Taniguchi and Urasawa,1987); incapacity of the sera to neutralize more than a few virus
particles (low titer antiserum; Narayan et al., 1984); generation ofneutralization-resistant mutant variants of the virus (Hussain et al.,
1987; Lambkin et al., 1994; Li et al., 1995; Watkins et al., 1993);
low afﬁnity of neutralizing antibodies, thus the need of long incubations to achieve effective neutralization (Torfason et al., 1992);
low stability of the antibody-virus complex, reversing easily after
dilution (Sune et al., 1990); and the presence of blocking antibodies
that inhibit virus neutralization (Massey and Schochetman, 1981;
O’Rourke et al., 1988).
Most of these hypotheses have been studied in ASF using
antisera from convalescent pigs infected with attenuated virus isolates and showing complete or incomplete virus neutralization(Gómez-Puertas and Escribano, 1997). Experiments revealed that
incomplete neutralization of ASFV can be caused by virus aggregation (Zsak et al., 1993) but is not caused by low afﬁnity or stabilityof virus-antibody complexes. Attempts to purify antigenic scapeComplex viruses commonly have more than one outer protein
which mediates neutralization, and this contributes to the complexity of neutralization (Dimmock, 1993). Often each protein has
multiple neutralization sites and probably different mechanismsof neutralization. In addition, neutralization efﬁciency may differ between proteins. A monoclonal antibody, mAb-135D4, thatrecognizes the major capsid protein p72 exhibited strong ASFV
neutralizing activity (Zsak et al., 1993). Consistent with the involvement of p72 in virus neutralization, immunoelectron microscopy,
using mAb-135D4, located this protein on the surface of nonenveloped virus particles (Borca et al., 1994). By analysis ofin vitro-translated products of the p72 gene speciﬁcally immunoprecipitated by mAb 135D4, a region between amino acid residues
400 and 404 was deﬁned as necessary for the reactivity of this mAb.
Five partially overlapping peptides (15mers) covering residues
388–446 failed to react with mAb 135D4, suggesting the conformational dependence of the epitope (Borca et al., 1994).
Sera from convalescent pigs revealed that p72, p30, and p54
were three of the most antigenic proteins during infection (Afonsoet al., 1992; Alcaraz et al., 1990, 1995; Gómez-Puertas et al., 1996;
Rodriguez et al., 1994, 1996). Pig immune sera against recombinant proteins p72, p30, and p54 neutralized more than 70% ofvirus infectivity (Gómez-Puertas et al., 1996). Surprisingly, antisera raised against recombinant protein p12 (Carrascosa et al.,1995) the viral attachment protein, did not reduce virus infectivity
in an in vitro assay (Gómez-Puertas et al., 1996). Immunoelectron microscopy of ultrathin sections of ASFV-infected Vero cells
using sera against these three proteins revealed their localizationin mature intracellular viral particles (Gómez-Puertas et al., 1996).
An analysis of the induction of the neutralizing antibodies mediated by these three proteins in the context of infection revealed
that afﬁnity-puriﬁed speciﬁc antibodies against these molecules,
generated during infection, neutralized the virus in a similar way
to monospeciﬁc antibodies obtained with recombinant proteins(Gómez-Puertas et al., 1996). Anti-p72 and -p54 sera neutralized
the virus only before attachment to susceptible cells. However,serum recognizing p30 neutralized the virus equally when antibodies were incubated with the virus before or after attachment to cells(Gómez-Puertas et al., 1996). Therefore, we may conclude that antibodies against p72 and p54 inhibit a ﬁrst step in the virus replication
cycle related to virus attachment, while anti-p30 antibodies blocka second step associated with virus internalization. These resultscorrelate with the two neutralization mechanisms observed with
sera from convalescent swine and allow the assignation of speciﬁc
proteins to these mechanisms.
The role of p54 and p30 proteins in receptor-mediated ASFV
endocytosis in swine macrophages has also been studied. These
two proteins, which are released from ASFV particles after treatment of virions with a non-ionic detergent, bound to virus-sensitive

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alveolar pig macrophages. This binding was found to be speciﬁcallyinhibited by neutralizing antibodies obtained from a convalescent
pig or from pigs immunized with recombinant p54 or p30 proteins (Gómez-Puertas et al., 1998). The binding of radiolabeled
recombinant p54 and p30 proteins to macrophages was speciﬁcally competed by an excess of unlabeled p54 and p30, respectively.
However, cross-binding inhibition was not observed, suggesting
the presence of two distinct saturable binding sites for these proteins in susceptible cells. In addition, p54 blocked the speciﬁc
binding of virus particles to the macrophage, while p30 impededvirus internalization. Both proteins independently prevented virus
infection and in a dose-dependent manner, thereby suggesting thatbinding interactions mediated by these two molecules are necessary to give rise to a productive infection (Gómez-Puertas et al.,1998).
The relevance of the blockade of virus–cell interactions by p54
and p30 in the protective immune response to ASFV has also beenaddressed. Immunization of pigs with either recombinant p54 or
p30 proteins induced neutralizing antibodies, which, as expected,
inhibited virus attachment and internalization, respectively. However, immunized pigs were not protected against lethal infection
and the course of the disease was not modiﬁed in these animals(only a delay in the disease onset was observed in p30-immunizedpigs). In contrast, immunization with a combination of p54 andp30 proteins simultaneously stimulated both virus-neutralizing
mechanisms and dramatically modiﬁed the course of the disease,rendering a variable degree of protection, ranging from a delayin the onset of the disease to complete protection against virus
infection (Barderas et al., 2001; Gómez-Puertas et al., 1998). In
an attempt to evaluate the role(s) of baculovirus-expressed p30,
p54, p72, and p22 proteins from another pathogenic ASFV isolate(Pr4) in protective immunity, other authors immunized pigs withthese proteins but failed to reproduce the degree of protection
obtained previously (Neilan et al., 2004). Although ASFV-speciﬁc
neutralizing antibodies were detected in the test group of animals,
these exhibited only a 2-day delay to onset of clinical disease andreduced viremia levels at 2 days post-infection. However, by day
4 there was no signiﬁcant difference in comparison to the nonimmunized control group and animals died between 7 and 10 days
after virus challenge. Differences in the immunization protocols orviruses used in experiments may determine the results obtained.
Additional research is required to establish optimal immunization
protocols and the true relevance of speciﬁc proteins in antibodymediated protection against ASFV.
Finally, the baculovirus expressed hemagglutinin of ASFV,showing hemadsorption and erythrocyte-agglutinating activities, characteristic of the CD2 homolog protein induced by thevirus in infected macrophages, triggered in immunized pigs
hemagglutination-inhibition and temporary infection-inhibition
antibodies. Interestingly, immunized pigs with this recombinant
protein were protected against lethal infection (Ruiz-Gonzalvo
et al., 1996). It opens new possibilities to incorporate more ASFV
proteins in a potentially effective subunit vaccine formulation.

7. Antibodies directed to the dynein-binding domain ofprotein p54 neutralize ASFVAt very early infection stages, ASFV interacts with the 8 kDalight chain of cytoplasmic dynein (DLC8), a microtubular-based
motor, through the structural virus protein p54. This interaction is
critical during virus internalization and transport to factory sites. A
13 amino acid (aa) domain of p54 has been shown to be sufﬁcientfor binding to DLC8 (Alonso et al., 2001). Since the p54 dyneinbinding domain (DBD) has been identiﬁed and peptides with thissequence have been successfully used to inhibit virus infection in

Fig. 5. Determination by ELISA of antibodies recognizing the dynein binding domain(DBD) of ASFV in sera from recovered pigs after infection (sera 6–9) or immunizedwith protein p54 (sera 2–5). ELISA plates were coated with puriﬁed E. coli-derived
p54 protein or with a peptide representing the DBD sequence.

cell cultures (Hernáez et al., 2010), we explored how antibodies
against this viral aa sequence interfere with ASFV infection.
In that study, we ﬁrst determined the immunological dominance of the DBD aa sequence in the context of
infection. The speciﬁc antibody response against the DBD
sequence was evaluated by ELISA, using synthetic peptides
representing the DBD of ASFV, in sera from pigs recovered fromexperimental virus inoculations. Additionally, we also analyzed
sera from pigs immunized with recombinant p54 (Fig. 5). Independently of the antibody titer of each serum analyzed, all sera
tested recognized the p54 protein, but only sera from recovered
pigs reacted in ELISA with the peptides belonging to the DBD. Thisﬁnding indicates that virus replication, which is normally required
to achieve protection against ASFV, is needed to generate speciﬁc
antibodies against the DBD sequence.
To test the potential of antibodies directed to DBD to inhibit
infection, we generated speciﬁc antibodies against this aa sequence.
Since peptides are usually of low immunogenicity, we fused theencoding sequence for DBD to Salmonella typhimurium type 2 (STF2)
ﬂagellin, a TLR5 ligand that promotes strong antibody responses inFig. 6. Expression of ﬂagellin fusion proteins containing the DBD ASFV sequenceby the baculovirus expression system. (A) Schematic representation of STF2, STF2DBD and STF2-4xDBD constructs cloned in a baculovirus vector. All 3 constructs
were designed with His-tag and KDEL sequence and were expressed in a baculovirus
expression system (Bac to Bac® ; Invitrogen). (B) Detection of the recombinant proteins STF2-DBD (2), STF2 (3), and STF2-4xDBD (4), produced in infected Sf21 insect
cells, by Western blot with a monoclonal antibody anti-His-tag. A protein extractfrom insect cells infected with the wild-type baculovirus without an insert was
included as a negative control (1). Arrows indicate the position of the recombinant proteins. (C) Puriﬁed recombinant proteins obtained by IBES® technology (T.
ni insects as living biofactories); STF2 (5), STF2-DBD (6), and STF2-4xDBD (7), wereobserved by Coomassie brilliant blue staining of SDS–PAGE gels.

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7Fig. 7. (A) Antibody immune response against several recombinant ﬂagellin-fusion proteins in immunized mice. The antibody response was measured by ELISA plates coatedwith puriﬁed E. coli-derived p54 protein. Sera were tested at a range of dilutions; Protein STF2 (C1 and C2), STF2-DBD (B1–B5) and STF2-4xDBD (A1–A5). Neutralization
values obtained with the sera from mice immunized with the ﬂagellin-DBD fusion construct using the virus isolate 1207 VR19 (B) or 608 VR14 (C). Neutralization analyses
were performed in Vero cells by a plaque number reduction assay using 100 PFU of the different viruses.

immunizations (Medzhitov et al., 1997). This strategy was successfully used previously to raise antibodies against this sequence asa peptide model (Terrón-Expósito et al., 2012). The ORF of STF2
protein was cloned into Pfastbac (Invitrogen) using Salmonella
typhimurium DNA as a template. The recombinant plasmidgenerated pFB-STF2-H-KDEL contained the KDEL sequence and 10
histidine residues, which facilitated the expression and puriﬁcation of the protein respectively. The DLC8 binding domain found in
the ASFV p54 protein was cloned into pFB-STF2-H-KDEL, in framewith STF2, to generate the pFB-STF2-DBD-H-KDEL plasmid. The
pFB-STF2-4xDBD-H-KDEL vector, encoding four copies of STF2, wasconstructed by cloning the PCR fragments in tandem into pFBSTF2-H-KDEL (Fig. 6A). In order to optimize protein expression, we
veriﬁed the production of recombinant proteins in sf21 cells prior
to the infection of Trichoplusia ni insect larvae (IBES® technology).
Total soluble protein fractions were extracted from insect larvaeand processed as described previously (Pérez-Filgueira et al., 2006).
Expression was analyzed by Western blot with a mAb against thehistidine tag. The antibody recognized speciﬁc bands corresponding to recombinant proteins STF2, STF2-DBD and STF2-4xDBD at
the expected electrophoretic mobility (Fig. 6B). Proteins obtained
from inoculated larvae were puriﬁed under native conditions andidentiﬁed in a Coomassie blue-stained gel (Fig. 6C).
Puriﬁed STF2-DBD fusion proteins were used to immunize mice.
Each mouse received 50 ␮g of protein per inoculation in Freund’s complete (ﬁrst dose) or incomplete adjuvant (second andthird dose). Fifteen days after the last injection, polyclonal serum
was prepared from deﬁbrinated blood and titrated by ELISA. The
immune response to the recombinant STF2-4xDBD was strong
in all animals (n = 5). In the case of the recombinant fusion protein containing one copy of the DBD sequence, the amount ofantibody produced varied from mouse to mouse, with 3 animals
with the same response than obtained with constructs containing four copies of DBD and 2 animals with a reduced response(Fig. 7A).
Sera from each group were tested for non-complementmediated virus neutralizing activity in Vero cell monolayers ina focus reduction neutralization test. Immune mice sera, raised
to one or four copies of the DBD, reduced the virus infection
plaques up to 61.5%, as determined by immunoﬂuorescence (Fig. 7B
and C). Foci, counted in duplicate wells, were averaged and compared to those obtained with neutralization assays performed
with pre-immune sera. Sera obtained against STF2 did not show
any neutralizing activity. On the basis of the above results, we
may conclude that the p54 DBD is implicated in virus neutralization mediated by antibodies. However, further experiments
are needed to determine how the induction of antibodies against
this domain modiﬁes the course of the disease in vaccinatedpigs.

8. Concluding remarks
Despite original reports indicating the lack of neutralizing
activity of sera from animals infected with ASFV, overwhelming
evidence of neutralizing antibodies against this virus has been
provided by numerous laboratories in the last 15 years. Moreover, several elegant experiments have revealed the relevanceof antibodies in protection against this fatal disease. However,in terms of antibody-mediated neutralization, ASFV has uncommon particularities that are shared by other viruses. Several ofthese singularities may explain why some authors have concluded
that ASFV does not induce neutralizing antibodies in pigs thathave recovered from infection. The peculiarities of ASFV include
loss of susceptibility to neutralization by cell culture passage as aresult of changes in the phospholipid composition of viral membranes and/or the presence of sera blocking antibodies that inhibit
complete neutralization. However, a number of ASFV proteins
have been undoubtedly implicated in the induction of neutralizing antibodies during infection and assigned to one of the twoneutralization mechanisms described for this virus. In addition,some critical epitopes in neutralization have also been characterized in proteins p72 and p54. Antibody-mediated neutralization
is a key defense mechanism against viral infections. Althoughcell-mediated immune mechanisms may make a signiﬁcant contribution to protection against ASFV, as in other viruses that infectmacrophages, current data encourage us to explore vaccine formulations with the aim to maximize the induction of critical andpotent neutralizing antibodies. Strategies to stimulate neutralizing antibodies should be considered feasible in the design of ASF
vaccine programs.
Acknowledgments
We thank Edel Reytor, Manuel Alonso and Miguel Andrade fortechnical assistance in protein expression by IBES® technology. The
novel data presented in this work were supported by the grant
CSD2006-00007-2 from the Ministry of Science and Innovation
and by funds from the company Alternative Gene Expression S.L.
(ALGENEX).
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Please cite this article in press as: Escribano, J.M., et al., Antibody-mediated neutralization of African swine fever virus: Myths and facts. Virus
Res. (2012), http://dx.doi.org/10.1016/j.virusres.2012.10.012