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Baldomero Cramer
Monoclonal antibodies can provide important pre- or post-exposure protection against infectious disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, lessened disease and improved survival in a controlled trial. Here, we present the cryo-EM structure at 3.1 Å of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with the antibodies in the Inmazeb cocktail. This structure allows the modeling of previously disordered portions of the glycoprotein glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities and residues critical for resistance to escape by these and other clinically relevant antibodies. We further provide direct evidence that Inmazeb protects against the rapid emergence of escape mutants, whereas monotherapies even against conserved epitopes do not, supporting the benefit of a cocktail versus a monotherapy approach.
In southern Italy, 44 contacts of hepatitis B virus carriers, including infants of carrier mothers, became HBsAg positive despite passive and active immunisation according to standard protocols. In 32 of these vaccinees infection was confirmed by the presence of additional markers of viral replication. In 1 infant, serious disease occurred. The virus from this patient is an escape mutant with a different sequence from that of the isolate from the mother. A point mutation from guanosine to adenosine at nucleotide position 587 resulted in an aminoacid substitution from glycine to arginine in the highly antigenic a determinant of HBsAg. This mutation is stable: it is present in an isolate from the child 5 years later. In some of these patients, including this child, the a determinant, to which a large part of the vaccine-induced immunity is directed, has been partly lost. Binding to HBsAg of a monoclonal antibody, previously mapped to the region of the mutation, was reduced in the child relative to that of the mother.
As far as B cells are concerned, the principal activity in this virus-centred scheme is mediated via the action of neutralizing antibodies, which inhibit infection of target cells. The action of neutralization may be simple, masking effects on binding molecules, but in other cases may involve opsonization of complement, Fc receptor binding, etc. Antibodies to internal or nonstructural viral components would be considered non-neutralizing (Zinkernagel 1996).
The closest human homologues of LCMV are arenaviruses, which tend to cause acute haemorrhagic disease, rather than persistence. However in the mouse, its natural host, it is relatively noncytopathic, and the various high level infections set up have been compared to human infection by HIV, HBV or HCV (Klenerman & Zinkernagel 1997). More recently established is the persistence of this virus at a low level over long periods. The biology of this and the mechanisms of immune evasion involved are much less well studied (Ciurea et al. 1999). The immune factors which control LCMV infection, and which influence at what level the virus persists have been established (Figs 5a, b). These are as follows:
Features that impair the maintenance of host CD8 + responses promote escape through speed. These include lack of CD4 T cells (Battegay et al. 1994); interestingly, lack of CD4 cells under some circumstances does not lead to complete exhaustion, but rather, maintenance of CD8+ T lymphocytes with reduced effector function (Zajac et al. 1998). It is likely that exposure of CD8+ T lymphocytes to high levels of antigen in circumstances where presentation is not on specialized DCs (i.e. in nonlymphoid organs) and without CD4 help, promotes antigen-induced cell death. Under similar conditions of prolonged antigenic exposure, CD4 exhaustion may also be observed, although the kinetics of induction are somewhat slower, perhaps because presentation is limited to Class II bearing cells (Oxenius et al. 1998; Ciurea et al. 2001).
In later stage HIV, HIV-specific CTL precursors disappear in preference to those specific for other infections (Carmichael et al. 1993). An inverse correlation has been observed between the numbers of HIV-specific CTL and viral load, which may indicate that high viral loads lead to suppression of such CTL (although the interpretation of this is complex) (Ogg et al. 1998). Also, early large expansions of CD8+ T lymphocytes bearing particularly restricted Vβ chains (i.e. of restricted specificity) has been associated with rapid progression, perhaps through a similar mechanism of activation-induced cell death (Pantaleo et al. 1994). On the other hand, T cells may persist, indeed individual clones may persist for many years in the face of continued viral replication (Kalams & Walker 1995). Also large expansions of activated T cells with maintained effector function are commonly seen in chronic HIV (Goulder et al. 2001), so if exhaustion occurs, it is not commonly the very rapid picture seen in experimental models. It is likely that it is one part of the ongoing dynamic interaction between host and virus.
In HBV and HCV, in contrast, high levels of virus are commonly associated with low levels of specific CD8+ T lymphocytes, and also low levels of specific CD4+ T lymphocytes (Guidotti et al. 1999; Lechner et al. 2000a Lechner et al. 2000a, b; Takaki et al. 2000). Evidence that high viral loads are associated with suppression of CD4+ T cells in HBV comes from studies where treatment with the antiviral drug lamivudine leads to emergence of specific CD4+ T cells, with similar data from HCV (although the treatment is more complex) (Ferrari et al. 1998; Cramp et al. 2000). In acute disease, CD8+ T cell responses are seen early, but may not be sustained, particularly in chronic HCV infection, as opposed to those who clear virus (Lechner et al. 2000a, b). Thus exhaustion of responses appears to be more likely in these infections.
How does this relate to human infection? Mutational escape of HIV from T cells (both CD8 and CD4) has been observed, in chronic infection (Phillips et al. 1991; Goulder et al. 1997; Harcourt et al. 1998) and, more rarely, during acute infection (Borrow et al. 1997; Price et al. 1997). In some cases, escape has been accompanied by clinical and virological deterioration, and this is confirmed by work in the SIV model (Dzuris et al. 2000; Vogel et al. 2001). Exactly what sort of immune responses lead to escape is by no means clear as the phenomenon is not universal. It is likely that focused responses are the most efficient at immune selection, but outside monkey models, information as to the breadth of the response, and, most importantly, the sequence of the infecting strain, is not always available.
Mutational escape has also been observed in HCV and HBV, but may occur less commonly than in HIV (Chang et al. 1997). Again, mutation leading to loss of T cell recognition has been observed in acute disease and in chronic disease (Chang et al. 1997), but clinical deterioration as a result of escape is not clearly reported. Part of the difficulty may be that of identifying the immunodominant response in any one individual at any one time. It should be pointed out that HBV is a DNA virus, but mutation may occur during the RNA intermediate phase, and particularly since replication rates are high. Drug resistance mutants, for example, emerge rapidly (Pichoud et al. 2000). Many more data are required before we can assess the role of shape-changing v speed or even camouflage in persistence of these infections.
The major strategy of MCMV to persist is to hide from the immune system by latency. After productive primary infection has eventually been cleared by the immune system the viral genome is not eliminated but persists as episomal DNA. In latency, viral proteins, which are the major target for the immune system, are not produced (or only to a very limited extent). From latent genomes productive replication and virus shedding can be reinitiated to allow virus transmission to a new host (Yu et al. 1995; Kurz & Reddehase 1999).Table 3
Herpes simplex virus (HSV) inhibits TAP transport using the protein ICP-47. In contrast to US6, ICP-47 affects peptide binding to TAP (Hill et al. 1995; Goldsmith et al. 1998), since it is recognized by TAP in a manner similar to peptides. Once bound, ICP-47 prevents peptide binding further by inducing conformational changes in the TAP heterodimer (Tomazin et al. 1996). MHC class I molecules, which are not loaded with translocated peptide, are retained in a tapasin-dependent manner in the ER lumen. These are at later stages targets for cytosolic relocation and proteasomal degradation.
The above molecules illustrate examples of modification of maturation, assembly and export of MHC-peptide complexes (Fig. 3). Other herpesviruses employ strategies to interfere with antigen processing at an earlier stage. EBV nuclear antigen (EBNA 1) is the dominant antigen expressed in latent EBV infection, yet is poorly processed when expressed intracellularly (Levitsky et al. 1997). This is due to its C-terminus containing a long glycine-alanine repeat, which renders the protein indigestible to proteasomes (Levitskaya et al. 1995). Interestingly, although these were missed in earlier studies, responses to this protein do occur, but in this case through cross-presentation (i.e. uptake by an uninfected DC and presentation through the Class I pathway) (Ferlazzo et al. 2000; Subklewe et al. 2001).
One of the main issues in this area is defining which of the phenomena are biologically relevant. This relies on an understanding not just of the biochemical processes involved in antigen processing or T cell recognition, but also on the physiology and anatomy of complex host immune responses. It is very easy to identify in vitro phenomena that influence T cell recognition, but often it is very difficult to demonstrate that such phenomena play an important role in vivo. Partly this may be because some effects are simply quantitative advantages in viral survival, which only become manifest over long periods in the host. This differs from, for example, drug escape strategies, which result from very intense selective pressures. The effect of the local environment, e.g. within salivary glands on the fine balance between host and pathogen requires more investigation.
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