Antiviral antibody responses: the two extremes of a wide spectrum Lars Hangartner, Rolf M. Zinkernagel and Hans Hengartner
Abstract | Viruses elicit a diverse spectrum of antiviral antibody responses. In this review, we discuss two widely used experimental model systems for viral infections — non-cytopathic lymphocytic choriomeningitis virus (LCMV) and acutely cytopathic vesicular stomatitis virus (VSV) — to analyse two fundamentally different types of antiviral antibody response. The basic principles found in these model infections are discussed in the context of other viral infections, and with regard to protective neutralizing versus non-protective enzyme-linked immunosorbent assay (ELISA)-detected antibody responses. Issues of antibody specificity, affinity and avidity, maturation and escape are discussed in the context of co-evolution of the host and viruses.
Latent Reversible dormant state of viruses in infected cells with minimal production of viral proteins and absence of progeny virus production.
Institute of Experimental Immunology, University Hospital Zurich, Schmelzbergstrasse 12, 8091 Zürich, Switzerland. Correspondence to H.H. e-mail: [email protected]
Viruses are obligate intracellular pathogens and therefore depend on living hosts for their propagation. In most cases, the co-evolution of virus and host has resulted in a balanced survival of both. This balance is usually the result of reduced pathological effects that are caused by the infecting virus, as well as increased nonspecific and specific immunological-resistance mechanisms by the host. Viruses that infect new host species, so-called ‘new’ or ‘emerging’ infections, are usually either much more pathogenic than better-adapted viruses (for example, the introduction of myxomatosis into Australian rabbits)1, or are not successful at establishing themselves within the general population (for example, Ebola virus). There is a broad spectrum of direct cytopathic effects caused by viruses. Acutely cytopathic viruses cause excessive damage in infected tissues and have to be controlled rapidly if the host is to survive. Examples of such cytopathic viruses in humans include the neurotropic poliovirus, rabies virus and smallpox virus. In mice, vesicular stomatitis virus (VSV) resembles rabies in humans and is used to model acutely cytopathic viruses (BOX 1). By contrast, other viruses do not induce direct cellular damage, and disease results from the ensuing immune response (immunopathology). Examples of such poorly or non-cytopathic viruses are hepatitis B virus (HBV), hepatitis C virus (HCV), and possibly HIV (see later) in humans, and lymphocytic choriomeningitis virus (LCMV) in mice (BOX 1). Poorly cytopathic viruses usually persist over the lifetime of the host, and are frequently transmitted from carrier mothers to their children through the placenta before birth (LCMV)2 or
NATURE REVIEWS | IMMUNOLOGY
at the time of birth (HBV, HCV and HIV)3,4. Persistence within a host can also be achieved by cytopathic viruses, but they require the ability to enter into latency within selected cell types to escape immunological responses. Moreover, many of these intermittently cytopathic viruses interfere with immune responses to prevent their clearance. Some prominent members of this group are herpes viruses, such as herpes simplex virus, cytomegalovirus and Epstein–Barr virus. They are widespread in the human population, persist life-long in sequestered cells and intermittently cause lytic infections5, 6. It is outside the scope of this review to discuss innate resistance to viral infections, such as the production of interferons. Nevertheless, it must be stated that without these mechanisms, the adaptive immune system, including antibodies, is not able to control virus infections7. Immune control of viruses usually requires a collaboration of the cellular and humoral arms of the immune system. In this review, we focus on LCMV and VSV as extreme examples of persisting, poorly cytopathic and acutely cytopathic virus–host interactions, respectively. Experimental findings made with these two viruses will be discussed in the context of other viral infections.
Types of antibody induced by viral infections Viral proteins are recognized as foreign (non-self), and antibodies against many viral proteins are raised following infection8,9. Yet, normally only a minor fraction of these antibodies have direct antiviral activity in vitro, and are therefore referred to as neutralizing antibodies. Neutralizing activity requires the antibody to be VOLUME 6 | MARCH 2006 | 231
REVIEWS Box 1 | Vesicular stomatitis virus and lymphocytic choriomeningitis virus Although cytopathic LCMV Viral titre vesicular stomatitis virus (non-cytopathic) ELISA-binding antibodies (VSV) and non-cytopathic Neutralizing IgG lymphocytic CTLs choriomeningitis virus (LCMV) represent two extreme cases within the general spectrum of virus– host relationships, we use them here as polar models to discuss general aspects of protective immune 0 4 8 12 16 60 responses. LCMV is a nonTime (days after infection) or poorly cytopathic arenavirus for which the mouse is the natural host. VSV Viral titre ELISA-binding antibodies (acutely cytopathic) Naturally, LCMV is Neutralizing IgG transmitted from virusCTLs carrier mothers to offspring Neutralizing IgM during pregnancy, reflecting a virtually optimal virus–host adaptation2. By contrast, VSV is an acutely cytopathic rhabdovirus that is closely related to 0 4 8 12 16 60 rabies virus. Its natural Time (days after infection) hosts are bovine species, in which infections cause vesicular stomatitis and, in rare cases, neurological symptoms. In the experimental mouse host, VSV exhibits exclusive neurotropism and, if not controlled by the immune response, causes paralytic disease about 5–8 days after infection, ascending up to the central nervous system, and finally causing death. VSV and LCMV elicit two distinct types of immune response in mice (see figure). Whereas prenatally transmitted LCMV persists in the host without priming immune responses, infections of adult mice induce cytotoxic-T-lymphocyte (CTL) responses that usually control the virus below detectable levels131,132. However, even after apparent control, LCMV might not be completely eliminated126. Neutralizing-antibody responses usually do not contribute during the acute phase of infection64,133 and only develop after 50–80 days134,135. However, if the CTL response is weak or absent, neutralizing antibodies become detectable earlier, around day 25–30 (REF. 32), and can suppress viremia successfully, albeit intermittently136. By contrast, the highly cytopathic VSV replicates only poorly in mice, except in neuronal cells, and is rapidly controlled by a strong, early, initially T-cellindependent, neutralizing IgM response that switches to a T-cell-dependent IgG response after 4–6 days137. Although virus-specific CTLs are induced during VSV infections, they are neither necessary nor sufficient to control infection. In fact, mice without CTLs control VSV as efficiently as CTL-competent mice138. ELISA, enzymelinked immunosorbent assay.
Complementaritydetermining region (CDR). The most variable parts of immunoglobulin molecules and T-cell receptors. These regions form loops that make contact with specific ligands. There are three such regions (CDR1, CDR2 and CDR3) contained in each V domain, with CDR3 arising from V(D)J rearrangement, and therefore being the most variable CDR.
of relatively high affinity and/or avidity for exposed structures on the surface of the virus10,11. Such antibodies render virions non-infectious by interfering with receptor binding and cell entry. However, amino-acid residues of viral surface proteins that are involved in receptor binding are frequently located in grooves that are not readily accessible to antibodies12–14. Neutralizing antibodies, therefore, frequently bind to structures that interfere with the interaction of the viral surface protein and its receptor by steric obstruction15,16. There are only a few human antibodies that have been characterized that contact the receptor-binding site directly12–14, and these do so through long complementarity-determining region 3
232 | MARCH 2006 | VOLUME 6
(CDR3) regions12. Some neutralizing antibodies were also shown to induce conformational changes that abrogate the functionality of the viral surface protein17. The process of viral entry into cells requires extensive conformational changes by viral surface proteins18,19, and binding of some neutralizing antibodies can therefore prevent viral entry by interfering with such conformational changes20,21. The vast majority of virus-specific antibodies, however, have no neutralizing activity. This is because they are elicited by virion fragments, or by viral proteins that are released from dying, infected cells22,23. Antibodies that are directed against such antigens are often specific for particular proteins: internal proteins that are not accessible on intact virions or infected cells (such as viral nucleoproteins)22,24; proteins that have been denatured, degraded or incompletely translated or processed (in terms of cleavage or glycosylation)23,25; or proteins that are not oligomerized (which is required for T-cell-independent activation of B cells; see below)25,26. Alternatively, antibodies can be elicited against native surface antigens, but do not have neutralizing activity because they are directed against epitopes for which antibody binding does not interfere with viral attachment or entry27. The protective function of such abundant antibodies is controversial. However, nonneutralizing antibodies that bind to surface-accessible determinants have been shown to help to control certain virus infections by activating the complement system, augmenting phagocytosis and/or promoting antibody-dependent cellular cytotoxicity. Antiviral antibodies can be detected either by their physical binding to viral components or by their biological function. Prototype assays used for the detection of antiviral antibodies are described in TABLE 1. For the detection of antibody binding to viral components, enzyme-linked immunosorbent assays (ELISAs) are frequently used. However, ELISA detection of antibodies normally gives no direct information regarding the biological function of the detected antibodies. This is because, on the one hand, only minimal affinities are required for detection by ELISA. On the other hand, immobilizing antigens on ELISA plates can alter the conformation of the proteins and therefore expose surfaces and epitopes that are not normally seen on intact virions. The biological function of antibodies is more complex to assess, and requires in vitro neutralization assays or in vivo protection assays to be carried out (TABLE 1). For mouse studies, both assays are feasible, and good correlations have been observed between the minimal in vitro neutralizing titre of the sera and in vivo protection. In the case of acutely cytopathic and intermittently cytolytic viruses, antibody kinetics that are detected by ELISA usually (but not always) correlate with in vitro neutralization titres28–31, and ELISA tests are therefore used to monitor the immune status of hosts. However, such a correlation does not readily apply to the persistencyprone, poorly cytopathic group of viruses, which induce ELISA-detectable antibodies early, whereas neutralizing antibodies are only detected weeks to months later32–36.
REVIEWS T-cell-independent antigen/ T-cell-dependent antigen Antigens that require specific T-cell help for eliciting antibody responses are designated as T-cell-dependent antigens. By contrast, T-cell-independent antigens elicit IgM antibody responses in the absence of specific T-cell help.
Epitope The part of an antigen that is directly recognized by antibodies or T-cell receptors.
The immunogenicity of intact viral surfaces Focusing on virions as a particulate antigen, there are three main factors that influence the antigenicity and immunogenicity of the native viral surface (FIG. 1). These are accessibility to antibodies, structural arrangement of the accessible site and frequency of germline-encoded immunoglobulin heavy-chain variable–light-chain variable (VH–VL)-region combinations with specificity for epitopes in the accessible sites. As illustrated in FIG. 1a, accessibility of viral surface antigens influences the number of antigenic sites against which antibodies can be raised. Some viruses, such as the enveloped and acutely cytopathic VSV, have densely packed surfaces that are composed of multiple copies of a single glycoprotein. Such dense packaging results in only a small area of the glycoprotein being accessible to the host viral receptor or to antibodies. As a consequence, intact VSV virions exhibit only one antigenic site, and antibodies that are directed against this are typically cross-competing and neutralizing10,37. Other viruses, such as poliovirus, have less densely
packed structures, often with protrusions at the surface (spikes). These spikes incorporate one or more proteins, with the antigenic sites typically located on apical loops that encompass the neutralizing epitope. As a result of this location, many viruses can alter the antigenic site without affecting the functionality of the protein19,38,39. Most viral surface proteins are present in a mulitmerized form on viral surfaces, often as trimers18. This implies that a considerable proportion of the monomeric surface is hidden inside the multimer, and is therefore not accessible to antibodies. Accessibility to antibodies can also be influenced by the presence of protein glycosylation. Additional glycosylation either can render antigenic sites inert19,34,40, or is necessary for the correct native conformation of the protein41. The arrangement of the antigenic sites on viral surface proteins has a great impact on the T-cell dependence of B-cell responses (FIG. 1b). Efficient, and therefore T-cell-independent, activation of B cells was originally characterized using haptenated polymers42 or polymeric flagellin43. Rigid two-dimensional arrays of about
Table 1 | Comparison of different assays for the analysis of anti-viral antibody responses Enzyme-linked immunosorbent assay (ELISA)
Samples to be tested are serially diluted and added to immobilized viral antigens on a plastic surface. Bound antibodies are revealed by a colour reaction for which the intensity can be quantified.
Infection of cultured cells with a viral inoculum that has been pre-incubated in the presence of serially diluted antibodies. Neutralizing titres are defined as the antibody dilution that reduces infectivity of the viral inoculum by 50% or more.
Following challenge of an actively or passively immunized animal, virus titres are determined in blood or organs and are compared with control animals that lack antiviral antibodies. Protection is considered to be sterile if all the viral inoculum is rendered inert immediately, and no signs of viral replication can be detected at any time point.*
In vitro or in vivo assay
Antigen used for detection
Purified viral proteins, cell lysates or peptides coated on plastic.
Infectious virus in suspension.
Infectious virus in suspension.
Conformation of antigen
Mostly denatured due to binding to plastic. If the antigen is captured by an antibody, the conformation is mostly native.
Native proteins, naturally oriented and oligomerized on the virion surface.
Native proteins, naturally oriented and oligomerized on the virion surface.
Random or fixed if antibody-captured.
Resulting epitope accessibility
Normally hidden or absent epitopes become accessible to antibodies. Capturing antibodies might cover naturally accessible epitopes.
Biologically relevant antibody– antigen interactions.
Biologically relevant antibody–antigen interactions.
Sum of all binding antibodies, relative concentration, specificity, avidity and isotype distribution.
Relative concentration of total or isotype-class-switched antiviral antibody activity.
Protective capacity of circulating antibodies.
Suitability for high ++++ throughput procedures
Information gained regarding protection
Fair correlation in the case of cytopathic viruses. No information in the case of noncytopathic viruses.
Good correlation between antibody titres measured and protection.
Direct assessment of protection. Protection is considered as sterile if there is no evidence of infection of host cells upon challenge.
* If no animal model is available, in vivo protection can be estimated statistically by correlating measured antibody levels with disease incidence or severity in large cohorts of individuals.
NATURE REVIEWS | IMMUNOLOGY
VOLUME 6 | MARCH 2006 | 233
REVIEWS a Accessibility
Single accessible antigenic site
Multiple accessible antigenic sites
Almost all Only a fraction surface-speciﬁc antibodies of surface-speciﬁc are neutralizing antibodies are neutralizing
Monomeric surface protein
Trimeric surface protein
Antibodies are induced against all surface areas
Some surface areas are not accessible to antibodies
Antibodies are induced against all apical areas
Glycosylation renders parts of the apical area antigenically inert
c B-cell repertoire
Random and/or mobile
T-cell-independent antibody production
T-cell-dependent antibody production
Areas not accessible to antibodies
Areas accessible to antibodies
High-affinity V regions frequently encoded in germline
High-affinity V regions rarely encoded in germline
Protective antibody response
Areas involved in viral entry
Poor antibody response
Non-accessible areas within the virion
Highlighted protein feature
Figure 1 | Factors influencing the immunogenicity of viral surfaces.
ELISA (Enzyme-linked immunosorbent assay). This assay can be used to detect antibodies that bind to an antigen, which is immobilized on a plastic surface. Samples to be tested are incubated on the coated plastic plates to allow binding of the contained antibodies to the coated antigen. Bound antibodies are then detected through an anti-immunoglobulin antibody coupled to an enzyme, which can catalyse a colour reaction.
Neutralization assay An in vitro assay used to detect direct antiviral activities of antibodies. Constant amounts of infectious virus are incubated with serially diluted antibodies and neutralizing titres are defined as the dilution that reduces infectivity in cell cultures by at least 50%.
30 haptens, optimally spaced by 5–10 nm, have been shown to induce B-cell responses in the absence of T-cell help. The neutralizing determinants of many cytopathic viruses (such as VSV) fulfill these structural requirements (see below). The frequency of germline-encoded immunoglobulin VH–VL-region combinations that are specific for accessible sites has a great impact on the immunogenicity of viral surfaces (FIG. 1c). For example, it has been shown that the neutralizing epitope of the LCMV surface glycoprotein is poorly immunogenic in wild-type mice44. By contrast, immunoglobulin-heavy-chain germline transgenic mice that express an increased frequency of B cells that are specific for the neutralizing epitope mount a potent, early and T-cell-independent antibody response, showing that the neutralizing epitope of LCMV is well presented to B cells45.
Antibody responses during viral infections Natural antibodies: first line of defense. The term ‘natural antibodies’ is used to describe low-affinity and polyreactive antibodies that are present at low titres in the blood of naive individuals. In mice, natural antibodies are present even under germ-free conditions46, and the self-renewing CD5+ B1 B-cell compartment has been
234 | MARCH 2006 | VOLUME 6
shown to secrete large amounts of these antibodies46,47. The polyreactive specificity of natural antibodies has been attributed to conformational flexibility within the CDR3 region of the immunoglobulin heavy chain, which allows these antibodies to bind to a broad range of antigens, including proteins, nucleotides, polysaccharides and lipids at low affinity48,49. Originally, natural antibodies were considered to be ‘nonspecific background’ in experiments, and most immunological assays were designed to exclude them. However, recent research has shown that natural antibodies provide an important link between the innate and adaptive immune systems by restricting initial viral dissemination50 (FIG. 2). Moreover, natural antibodies contribute substantially to the recruitment of viral antigens to secondary lymphoid organs, which is a prerequisite for the priming of adaptive immune responses51. Part of this process might be mediated by the recently discovered Fcα/µ receptor52, but complement that is contained in immune complexes that bind to complement receptors has also been described to contribute substantially to antigen recruitment50,53. In addition, complement receptor-2 is a signal-enhancing component of the B-cell receptor that facilitates B-cell activation54.
REVIEWS Limitation of initial viral dissemination
Marginal/sinusoidal zone of lymphoid organs
Priming of the adaptive immune system
Direct neutralization Filtering
Antigen recruitment to secondary lymphoid organs
Complement receptor 3 and 4 Complement (C1q, C3)
Activation of the complement system
Complement receptor 2 B-cell receptor
Binding to complement receptors leading to removal of immunecomplexed virus from circulation
Complement-mediated lysis of virions or infected cells
For example, T-cell-independent activation of neutralizing B cells
Figure 2 | Importance of natural antibodies. Natural antibodies provide an important link between the innate and adaptive immune systems. Before the adaptive immune system is activated, they restrict viral dissemination by direct neutralization, complement activation and elimination of virus in the marginal/sinusoidal zone of secondary lymphoid organs. Moreover, natural antibodies favour priming of the adaptive immune system by contributing substantially to antigen recruitment in secondary lymphoid organs.
Hapten A small molecule, or part of a molecule, that can elicit antibody responses when it is chemically linked to a carrier, but that is not immunogenic by itself. B-cell responses against haptens require priming of T-helper cells that are specific for the carrier, unless they are repetitively linked to a rigid carrier at a distance of 5–10 nm.
Some natural antibodies can neutralize viruses directly. Spontaneous pre-immune neutralizing antibody titres can be relatively high for cytopathic viruses (for example, 1:8 to 1:32 against VSV)45,55,56, but are generally low or below detection levels for poorly cytopathic viruses (for example,