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Journal of General Virology (1993), 74, 2039-2045.

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Review article New observations on antigenic diversification of RNA viruses. Antigenic variation is not dependent on immune selection Esteban Domingo,* Juana Diez, Miguel A. Martinez,'~ Javier Herndndez, Africa Holguin, Bel~n Borrego and Mauricio G. Mateu Centro de Biologia Molecular "Severo Ochoa' (CSIC-UAM), Universidad Auttnoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Introduction Antigenic variation amongst viruses arises as the result of amino acid substitutions in the viral protein domains that interact with components of the immune system. Point mutations, deletions and additions, as well as the entire replacement of genes or genomic segments by recombination or reassortment (in segmented viral genomes) may lead to viruses with altered antigenic specificities. In viral populations variants resistant to a neutralizing monoclonal antibody (MAb) are found at frequencies in the range of 10-3 to 10-~ mutants per infectious genome (for reviews see Yewdell & Gerhard, 1981; Domingo & Holland, 1988). However, it is important to understand how variants present in such a low proportion become dominant in virus populations evolving in the course of disease progression or during epidemic outbreaks. Also, the rules, if any, on the number and nature of amino acid substitutions needed to endow viruses with new antigenic specificities are extremely relevant to disease prevention by vaccination. In this report we approach these questions in the light of the quasispecies structure of RNA viruses. Results from studies with field isolates of viruses and analysis of virus populations passaged in cell culture suggest novel features in the process of antigenic diversification.

Genetic variation of viruses as a two-step process The genetic variation in RNA viruses is the result of at least two distinct processes. First is the generation of mutant genomes during viral replication which is an inevitable consequence of the error-prone nature of template copying by RNA polymerases and retroviral reverse transcriptases (Holland et al., 1982, 1992). The tPresent address: Laboratoire de Rttrovirologie Moltcutaire, Institut Pasteur, 75724 Paris C~dex 15, France. 0001-1579 © 1993 SGM

parameter that determines the generation of mutants is the mutation rate, defined as the number of misincorporation events per nucleotide site per round of copying. For at least a substantial number of genomic residues of the RNA viruses examined, mutation rates are in the range of 10 3 to 10-5 substitutions per site per round of copying. However, a particular type of mutation may be favoured or impaired by influences such as the nature of the viral enzyme under study, sequence context of the site, concentration of nucleotide pools in the cell, etc. (recent review in Holland et al., 1992). Mutation rates at such high levels ensure that many mutants arise and are present in each infected cell (and obviously in any infected tissue or entire organism). This has been proven by using the PCR amplification of viral genomes from infected tissues, avoiding adaptation of viruses to cell culture (Meyerhans et al., 1989; Martell et al., 1992; Benmansour et aI., 1992; for review see Domingo & Holland, 1993). A mutation rate refers to a biochemical event during polymerase activity and must be distinguished from mutation (or mutant) frequency which is the proportion that a given mutant attains in a virus population. This proportion depends not only on the rate at which the mutant arises but also on the result of competition between the mutant and all other variants. The competitive ability of a virus is the result of the efficacy of any biochemical process in the virus life cycle: genome replication, protein synthesis and processing, particle assembly, release from cells, stability of particles outside the cell, etc. Following the competition between variant genomes, long-term evolution will be additionally influenced by positive selection forces and random sampling events such as transmission of one or a few infectious particles from an infected into a susceptible host (for review see Domingo & Holland, 1993). In the competition established immediately following the generation of variants, a newly arising mutant will never dominate over the initial virus distribution if the latter is better adapted to

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E. D o m i n g o and others

the environment in which replication is taking place. This situation in which a dynamic spectrum of mutants produces the same average or consensus sequence repeatedly is termed' population equilibrium', and it has been experimentally documented, among other systems, for phage Q8 (Domingo et al., 1978), vesicular stomatitis virus (Holland et aI., 1982; Steinhauer et al., 1989) and a number of plant viruses (van Vloten-Doting & Bol, 1988). In spite of being in equilibrium, an R N A virus population exists as a distribution of mutant genomes (sustained by input of mutants during genome replication) tei3ned viral quasispecies. A quasispecies may include one or several dominant, master sequences which very often represent a minority in the population of molecules (recent reviews on viral quasispecies in Holland et al., 1992; Domingo et al., 1993). This population equilibrium is a type of metastable equilibrium which will be perturbed as soon as mutants with higher competitive fitness than the previous ensemble arise as a result of the stochastic process of mutation. An alternative way to induce disequilibrium is through an environmental change such that a component of the mutant spectrum has a higher fitness than that of the previously dominant genomes. No matter how high mutation rates might be, in a situation of population equilibrium no evolution will take place. This two-step process (mutant generation followed by competitive rating) of genetic variation has an important influence on the antigenic diversification of RNA viruses.

Emergence and dominance of antigenic variants in the absence of immune selection Antigenic variation is extensive among viruses as diverse as influenza viruses (Webster et al., 1982; Palese & Young, 1982; Both et al., 1983; Oxford et al., 1986; Skehel & Wiley, 1988), lentiviruses (Kono et al., 1973; Montelaro et al., 1984; Clements et al., 1988; Burns & Desrosiers, 1991 ; Goudsmit et at., 1991), hepadnaviruses (Carman et al., t990) and picornaviruses (Hovi et al., 1986; Minor et al., 1986; Gebauer et aL, 1988; Carrillo et al., 1990). It has been proposed (and in many cases assumed) that antigenic variation of viruses is the result of direct selection of variants by the host immune humoral and/or cellular responses. However, an early study by Hampar & Keehn (1967) documented a change in the antigenic specificity of herpes simplex virus in the course of a persistent infection in cell culture in the absence of antiviral antibodies. Similarly, BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) led to a gradual change in antigenic properties of the resident virus, as revealed by its altered reactivity with MAbs (Diez et al., 1990). The emergence of antigenic variants of FMDV was not observed only in

the course of persistence, since a variant of FMDV A22 Iraq 24/64 became dominant in the population when the parental virus was adapted to growing in spinner suspension cultures of BHK-21 cells (Bolwell et al., 1989). Serial passage of FMDV in BHK-21 cell monolayers resulted also in the emergence and dominance of antigenic variants without any involvement of immune selection (Diez et al., 1989; Martinez et al., 1991a). Similarly, a cytopathic variant of hepatitis A virus (strain HM-175) became dominant after about 20 passages of persistently infected BS-C-1 cells (Cromeans et al., 1989). This virus, and several clonal derivatives from the same populations, had acquired a substitution within an immunodominant neutralization site :in the absence of selective antibody pressure (Lemon et al., 1991). Lentiviruses cause slow, chronic and progressive disease in man and animals. The role of immune selection in the maintenance of lentivirus persistence (Kono et al., 1973; Montelaro et al., 1984; Clements et aI., 1988; Burns & Desrosiers, 1991) was disputed by some authors (Thormar et al., 1983; Lutley et al., 1983). Studies by Carpenter et al. (1987, 1990) on the variation in equine infectious anaemia virus revealed that the sequential appearance of two antigenic variants was independent of the development of variant-specific neutralizing antibody. The authors suggested that selection of variants could occur via elimination of infected cells (by antibodyor cell-mediated cytotoxicity) prior to an increase in neutralizing antibody. Alternatively, the variant-specific immune response might be a reactive phenomenon secondary to the sequential appearance of viral variants selected by other biological mechanisms, as suggested by Carpenter et al. (1990). Serial passage of clones of human immunodeficiency virus type 1 (HIV-1) in cell culture in the absence of anti-HIV-1 antibodies resulted in amino acid substitutions at the antigenic loop V3 (S~inchezPalomino et al., 1993). Thus, there is considerable evidence that lentiviruses may undergo antigenic variation in the absence of immune selection. The internal proteins of several paramyxoviruses have undergone a degree of variation similar to that of the external glycoproteins that elicit neutralizing antibodies (Sato et al., 1985; Rydbeck et al., 1986). In Newcastle disease virus (NDV), the external haemagglutininneuraminidase did not differ significantly from either the membrane protein or from one of the polymerase proteins in its degree of antigenic variation (Nishikawa et al., t987). Results from studies of the reactivity of field strains with four groups of MAbs directed against each of the proteins compared led to the suggestion that spontaneous mutation rather than immunological selection pressure is responsible for the antigenic variation of NDV (Nishikawa et al., 1987; for review see Russell et al., 1990).

R e v i e w : R N A virus antigenic diversification

In spite of considerable evidence for positive immune selection occurring in the antigenic diversification of influenza viruses, some studies suggest the fixation of antigenically relevant substitutions by other mechanisms. Influenza B virus subpopulations showing different cell tropisms were also antigenically distinct (Schild et al., 1983). Two variants of swine influenza virus (H1NI) termed H (high-yielding) and L (low-yielding) differed in replication properties as well as in antigenicity (Both et al., 1983). The phenotypic conversion from L to H was associated with an amino acid replacement [Gly (155) -~ Glu] at an antigenic site of the haemagglutinin (HA). The analysis of revertant viruses indicated that this substitution affected replication properties as well as antigenicity, thus providing evidence for fortuitous antigenic change in association with change in biological function (Both et al., 1983). However, an antigenic site need not change only because the residues involved perform a second function related to viral replication. Rocha et al. (1991) sequenced the HA and nucleoprotein genes of sequential isolates of influenza virus type A (H1N1) from a child with severe combined immunodeficiency syndrome who was persistently infected with influenza virus. Multiple, non-cumulative mutations were identified in those viral genes over a period of several months. About half of the mutations in the R N A encoding the HA1 domain, which includes the five main antigenic sites, led to an amino acid substitution. All the observed replacements were located at (or very close to) antigenic sites. Thus, antigenic variants emerged at high frequency despite a diminished (or absent) immune response. Rabies virus was considered monotypic until MAbs revealed considerable variation in the epitopes of its N and G proteins (Wiktor & Koprowski, 1978; Smith et al., 1986). However, viruses isolated within a disease outbreak were regarded as genetically and antigenically stable (Smith, 1989; for review see Baer et al., 1990). A recent study by Flamand and associates has clearly documented the quasispecies structure of rabies virus by revealing at least seven distinct sequences in a glycoprotein domain of virus isolated from the brain of a rabid dog (Benmansour et al., 1992). Furthermore, they documented the selection of an antigenic variant from a human isolate that was adapted for better growth in cell culture. Again, antigenic variation of rabies virus was not necessarily the result of immune selection (Benmansour et al., 1992).

Random change model for antigenic variation of viruses: negative versus positive selection There are at least two possible mechanisms to explain the eventual dominance of antigenic variants in viruses

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evolving in the absence of immune pressure. Firstly, the variants may result from selective forces unrelated to an immune response but which may favour amino acid replacements that result in a change of antigenic specificity. As discussed above, this mechanism may account for the change of antigenicity observed in swine influenza virus due to an amino acid substitution which may also improve virus-cell receptor interaction (Both et al., 1983; Kilbourne et aL, 1988). Likewise, the frequent occurrence of antigenic variants of hepatitis A virus in cell culture, which contrasts with a more limited variation among wild-type human strains, suggests that there might be a natural restriction of substitutions at some antigenic sites due to their possible involvement in virus attachment and entry (Lemon et al., 1991). Antigenic sites of picornaviruses often surround domains of attachment to cells (Rossmann & Johnson, 1989; Acharya et al., 1989; Scraba, 1990). In FMDV a highly conserved VP1 sequence (Arg-Gly-Asp, also found in some cell attachment proteins), which is located within a major antigenic site, may be part of the receptor recognition domain (Fox et al., 1989). Thus, substitutions around this conserved amino acid triplet, fixed in the absence of immune selection, may affect binding to cells as well as antigenic specificity (Diez et al., 1989; Marfinez et al., 1991a). However, our attempts to demonstrate differences in binding to cells among FMDVs harbouring substitutions around the Arg-GlyAsp sequence have been as yet unsuccessful (A. Holguin, unpublished). The second model that explains antigenic variation in the absence of immune selection, which has been generally neglected previously, is based on the population dynamics imposed by the quasispecies structure of RNA viruses. Some aspects of this model have been discussed by Diez et al. (1989, 1990), Rocha et al. (1991) and Domingo et al. (1992). As described above, competitive selection is continuously (and unavoidably) acting on replicating genomes. Mutants with a selective disadvantage will be kept at low levels (or will be eliminated) by negative selection. As a consequence, viral quasispecies will be populated mostly by variants that behave in the most neutral way in this environment (Temin, 1989; Domingo et aL, 1992). We suggest that since antigenic sites are located on the surface of capsid or envelope proteins, they will be subjected to less stringent structural requirements than domains involved in interactions with other structural proteins. Thus, fluctuations in genomic distributions, promoted either by an environmental change, by the stochastic generation of mutants more fit than their parental counterparts, or by random sampling events (bottleneck transmission), have a certain probability of incorporating as accompanying mutations those which are more neutral (tolerated by

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E. Domingo and others

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it must be emphasized that the spectrum of mutants of a viral quasispecies will often be shaped by competitive selection among individual genomes. Furthermore, positive selection driven by mutations occurring anywhere in the genome, not only random sampling events, contributes to fixation of amino acid replacements at antigen sites. Positive selection may also be driven by replacements occurring at antigenic sites due to their involvement in other biological functions, as documented in the previous section. A scheme of the random change model of antigenic variation is shown in Fig. 1. [See Domingo & Holland (1993) for additional discussion on the selectionist versus neutralist models as they apply to RNA virus evolution.]

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Fig. 1. A random change model for antigenic variation of viruses in the absence of immune selection. Lines represent genomic R N A molecules; A.S. is an R N A region encoding an antigenic site. Symbols on the lines represent mutations which accumulate within A.S. where mutations are more tolerated. (a) In spite of increased frequency of substitutions within A.S. no modification o f the consensus sequence is observed because a large number o f molecules replicate to provide progeny populations (wide arrows). (b) When the same mutant spectrum is subjected to a random sampling event or to a positive selection event (small arrow) a change in the consensus sequence is observed, involving a preferential fixation of replacements within A.S. It must be noted that positive selection may be mediated either by one of the replacements within A.S., arising from its involvement in biological roles other than antigenicity, or by a replacement elsewhere in the genome (circle in the selected genome). The model explains fixation o f replacements at any genomic site where replacements are tolerated more than average in the viral genome.

negative selection). Thus, we suggest that the random occurrence of tolerated replacements is a mechanism of antigenic diversification of highly variable RNA viruses, operating in addition to direct positive selection by the host immune response. We coined the term 'random change model' for this new mechanism of antigenic diversification. It is important to note that this mechanism necessitates the action of negative selection on continuously arising new variants and, thus, the mechanism depends on the quasispecies structure of R N A viruses. Although the model we propose has features in common with the neutral theory of molecular evolution,

The effect of single amino acid replacements may be compensated by additional substitutions Variant viruses with a single amino acid replacement at an exposed capsid site are likely to be well represented in quasispecies distributions since the latter are essentially the result of the stochastic generation of mutants and their rating by competitive selection (Eigen & Schuster, 1979; Domingo et al., 1985; Eigen & Biebricher, 1988). Naturally occurring single amino acid replacements may have a drastic effect on antigenic specificity (Mateu et al., 1989, 1990; Martinez et al., 1991 b; Wolfs et al., 1991). In some cases the effect of one replacement is enhanced by a second replacement at the same epitope. However, the effect of a critical substitution may be suppressed by additional replacements, themselves exerting no noticeable antigenic modifications (Mateu et al., 1992). A non-additive effect of multiple amino acid substitutions on antigenicity was first observed by Boyer et al. (1990) on the recognition of a T cell epitope in a cellular protein. This non-additivity of the effects of multiple replacements may help to keep viruses within antigenic groups by limiting their linear antigenic diversification. It is very likely that such effects, which introduce considerable uncertainty regarding the extent of antigenic evolution, will often be found both for B cell and T cell epitopes of highly variable RNA viruses because of the ample opportunity for multiple replacements at individual epitopes and antigenic sites.

Antigenic variation of F M D V type C does not require linear accumulation of amino acid substitutions over prolonged time periods: implications for a quasispecies genome organization A phylogenetic tree for FMDV isolates of serotype C spanning 6 decades has revealed that the evolution of capsid genes was due to accumulation of silent muta-

Review: R N A virus antigenic diversification tions, but that linear accumulation of amino acid replacements with time was not statistically significant (Martinez et at., 1992). The antigenic diversification of FMDV type C was due to changes in a limited number (often two) of amino acids at the most variable positions. It is not possible at present to assess whether antigenic diversity without net accumulation of amino acid substitutions is a widespread phenomenon in the longterm evolution of viruses. There are only a few viruses with isolates available in sufficient number (spanning several decades) that allow reliable phylogenetic trees to be constructed and mutations to be numerically analysed. In the case of influenza virus, net accumulation of amino acid substitutions occurs, even though the rates are several times lower than those for silent mutations (Bean et al., 1992 and references therein). The non-cumulative nature of amino acid substitutions at antigenic sites reinforces the effect that a quasispecies genetic organization has on promoting changes in antigenic behaviour. If variations among related sequences are sufficient to alter antigenicity, antigenic changes will occur as a result of alterations in population equilibrium, as discussed above. If entirely new amino acid sequences were often needed, correspondingly large jumps in the quasispecies distributions would also be required, and a viral system could exhaust its potential for variation and adaptability. This brief review emphasizes the importance of variations in related amino acid sequences (or quasispecies distributions) in promoting antigenic change. Results obtained with FMDV agree with observations on a number of RNA viruses, and show the indeterminate nature and unpredictability of behaviour at a molecular level of RNA virus populations (Holland et al., 1982, 1992).

Summary Recent results have revealed novel features in the process of antigenic diversification of FMDV. (i) Antigenic variation is not necessarily the result of immune selection. (ii) Single, critical amino acid replacements may either have a minor effect on antigenic specificity or cause a drastic antigenic change affecting many epitopes on an antigenic site. (iii) The effect of such a critical replacement may be suppressed by additional substitutions at neighbouring sites. (iv) Antigenic diversification does not necessarily involve net accumulation of amino acid substitutions over time. We review evidence that some of these features apply also to other riboviruses and retroviruses. A model is proposed to relate antigenic variation without immune selection to the quasispecies structure of RNA virus populations.

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Work supported by DGICYT n. PB91-0051-CO2-01, EC and Fundacirn Ram6n Areces. J. Hernfindez and A. Holgufn are predoctoral fellows from Comunidad Autrnoma de Madrid and B. Borrego from Ministerio de Educacirn y Ciencia. M. A. Martinez was supported by a postdoctoral fellowship from the Spanish Research Council.

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