Antigenic Escape Research Articles

Mutational analysis of a principal neutralization domain of visna/maedi virus envelope glycoprotein

We have shown previously that a type-specific neutralization domain is located within a 39 aa sequence in the fourth variable domain of gp135 in visna/maedi virus. We now show that neutralizing antibodies detected early in infection are directed to this epitope, suggesting an immunodominant nature of this domain. Ten antigenic variants were previously analysed for mutations in this region, and all but one were found to be mutated. To assess the importance of these mutations in replication and neutralization, we reconstructed several of the mutations in an infectious molecular clone and tested the resulting viruses for neutralization phenotype and replication. Mutation of a conserved cysteine was shown to alter the neutralization epitope, whilst the replication kinetics in macrophages were unchanged. Mutations modulating potential glycosylation sites were found in seven of the ten antigenic variants. A frequently occurring mutation, removing a potential glycosylation site, had no effect on its own on the neutralization phenotype of the virus. However, adding an extra potential glycosylation site in the region resulted in antigenic escape. The results indicate that the conserved cysteine plays a role in the structure of the epitope and that glycosylation may shield the principal neutralization site.

Structural basis of antigenic escape of a malaria vaccine candidate

Antibodies against the malaria vaccine candidate apical membrane antigen-1 (AMA-1) can inhibit invasion of merozoites into RBC, but antigenic diversity can compromise vaccine efficacy. We hypothesize that polymorphic sites located within inhibitory epitopes function as antigenic escape residues (AER). By using an in vitro model of antigenic escape, the inhibitory contribution of 24 polymorphic sites of the 3D7 AMA-1 vaccine was determined. An AER cluster of 13 polymorphisms, located within domain 1, had the highest inhibitory contribution. Within this AER cluster, antibodies primarily targeted five polymorphic residues situated on an alpha-helical loop. A second important AER cluster was localized to domain 2. Domain 3 polymorphisms enhanced the inhibitory contribution of the domain 2 AER cluster. Importantly, the AER clusters could be split, such that chimeras containing domain 1 of FVO and domain 2 + 3 of 3D7 generated antisera that showed similarly high level inhibition of the two vaccine strains. Antibodies to this chimeric protein also inhibited unrelated strains of the parasite. Interstrain AER chimeras can be a way to incorporate inhibitory epitopes of two AMA-1 strains into a single protein. The AER clusters map in close proximity to conserved structural elements: the hydrophobic trough and the C-terminal proteolytic processing site. This finding led us to hypothesize that a conserved structural basis of antigenic escape from anti-AMA-1 exists. Genotyping high-impact AER may be useful for classifying AMA-1 strains into inhibition groups and to detect allelic effects of an AMA-1 vaccine in the field.

Barriers to antigenic escape by pathogens: trade-off between reproductive rate and antigenic mutability

BackgroundA single measles vaccination provides lifelong protection. No antigenic variants that escape immunity have been observed. By contrast, influenza continually evolves new antigenic variants, and the vaccine has to be updated frequently with new strains. Both measles and influenza are RNA viruses with high mutation rates, so the mutation rate alone cannot explain the differences in antigenic variability.ResultsWe develop a new hypothesis to explain antigenic stasis versus change. We first note that the antigenically static viruses tend to have high reproductive rates and to concentrate infection in children, whereas antigenically variable viruses such as influenza tend to spread more widely across age classes. We argue that, for pathogens in a naive host population that spread more rapidly in younger individuals than in older individuals, natural selection weights more heavily a rise in reproductive rate. By contrast, pathogens that spread more readily among older individuals gain more by antigenic escape, so natural selection weights more heavily antigenic mutability.ConclusionThese divergent selective pressures on reproductive rate and antigenic mutability may explain some of the observed differences between pathogens in age-class bias, reproductive rate, and antigenic variation.

Genome Rearrangements, Deletions, and Amplifications in the Natural Population of Bartonella henselae

Cats are the natural host for Bartonella henselae, an opportunistic human pathogen and the agent of cat scratch disease. Here, we have analyzed the natural variation in gene content and genome structure of 38 Bartonella henselae strains isolated from cats and humans by comparative genome hybridizations to microarrays and probe hybridizations to pulsed-field gel electrophoresis (PFGE) blots. The variation in gene content was modest and confined to the prophage and the genomic islands, whereas the PFGE analyses indicated extensive rearrangements across the terminus of replication with breakpoints in areas of the genomic islands. We observed no difference in gene content or structure between feline and human strains. Rather, the results suggest multiple sources of human infection from feline B. henselae strains of diverse genotypes. Additionally, the microarray hybridizations revealed DNA amplification in some strains in the so-called chromosome II-like region. The amplified segments were centered at a position corresponding to a putative phage replication initiation site and increased in size with the duration of cultivation. We hypothesize that the variable gene pool in the B. henselae population plays an important role in the establishment of long-term persistent infection in the natural host by promoting antigenic variation and escape from the host immune response.

Identification of genetic diversity by cultivating influenza A(H3N2) virus in vitro in the presence of post-infection sera from small children

Antigenic variants probably arise in the field by escaping herd immunity. We have earlier found that sera from small children are more strain-specific than sera from adults and could therefore, provide favourable conditions for selecting antigenic escape mutants. We had access to small volumes of anonymous sera collected in Norway after the epidemic season 1999/00, which was dominated by the A/Panama/2007/99 (H3N2) variant. The HA gene of the representative strain of that season was genetically identical to A/South Australia/147/99 (H3N2) and was selected for this study. Two sera from children aged 4 and 3 years, respectively, and one adult (64 years old) were used to attempt selecting antigenic escape mutants. Virus was grown in MDCK cells in the presence of human serum and escaped variants were tested by haemagglutination-inhibition tests. Although variant strains were occasionally identified, their HA1 genetic sequence did not identify obvious changes at known antigenic sites. However, by cloning and subsequent sequencing, the genetic diversity of the parent virus was found to be significantly reduced when grown in the presence of human sera. Data also showed that the two children's sera selected additional mutants from those already present in the parent pool and that the two sera selected different mutants. On a community level, it is possible that antigenic changes could be accumulated in a step-wise manner when epidemic virus is transmitted from one small child to the next, each with a restricted and possibly variant antibody repertoire.

Mathematical models of immune effector responses to viral infections: Virus control versus the development of pathology

This article reviews mathematical models which have investigated the importance of lytic and non-lytic immune responses for the control of viral infections. Lytic immune responses fight the virus by killing infected cells, while non-lytic immune responses fight the virus by inhibiting viral replication while leaving the infected cell alive. The models suggest which types or combinations of immune responses are required to resolve infections which vary in their characteristics, such as the rate of viral replication and the rate of virus-induced target cell death. This framework is then applied to persistent infections and viral evolution. It is investigated how viral evolution and antigenic escape can influence the relative balance of lytic and non-lytic responses over time, and how this might correlate with the transition from an asymptomatic infection to pathology. This is discussed in the specific context of hepatitis C virus infection.

Antibody Neutralization Escape Mediated by Point Mutations in the Intracytoplasmic Tail of Human Immunodeficiency Virus Type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.

Resistance of HIV-infected cells to cytotoxic T lymphocytes

Although cytotoxic T lymphocytes (CTLs) are important for controlling HIV, CTLs are not effective at eradicating HIV infection. Recent studies have revealed a combination of factors that together make HIV-infected cells resistant to CTLs and make anti-HIV CTLs ineffective. These factors likely contribute to prolonged survival of infected target cells, which in turn increases the probability of antigenic variation and immune escape.

Influenza H5 virus escape mutants: immune protection and antibody production in mice

Avian H5N1 influenza A viruses are considered to be of high pandemic potential as they are able to cross the avian-human species barrier and cause disease in humans. In the present study we assessed the impact of amino acid substitutions in the hemagglutinin (HA) of antigenic escape mutants of influenza A/Mallard/Pennsylvania/10218/84 (H5N2) (Mld/PA/84-MA) virus on the level of neutralizing antibodies and the ability to protect mice against challenge with the wild type H5 influenza virus. β-Propiolactone-inactivated vaccines prepared from eight different H5 escape mutants could be separated into two groups based on levels of protection. One group of escape mutants [m46(7), m46(7)-24B9, m46(7)-55, and m46(7)-55-24B9] was characterized by providing high levels of protection (90.0–95.4% survival) to mice against subsequent challenge with 5 LD 50 of wild type Mld/PA/84-MA virus. The other group of escape mutants [m176/26, m55(2), m55(2)-24B9, and m24B9-176/26] provided moderate level of protection (57.1–66.6% survival) in mice. Analysis of the amino acid substitutions in the HA revealed that two amino acid changes in antigenic site B of the HA molecule ( D 126→ N and K 152→ N) were associated for decreases in the levels of antibody and the immune protection afforded by vaccination with these H5 virus escape mutants. The phenotypic effects of mutations in HA gene of H5 virus may be of importance to appraise the extent and direction of H5 influenza viruses antigenic evolution.

Hepatitis C virus dynamics and pathology: the role of CTL and antibody responses

This paper investigates the role of CTL and antibody responses in hepatitis C virus (HCV) dynamics and pathology. Mathematical models suggest that a strong CTL response is required for resolution of HCV infection and that a weak CTL response can result in persistent infection. According to the model, establishment of persistent infection is accompanied mainly by an ongoing antibody response, while CTLs are not maintained at high levels. In the model, this outcome correlates with absence of pathology. Persistent infection in the face of an ongoing antibody response can result in evolution of antigenic escape. According to the model, evolution towards escape from antibodies can shift the balance of immune responses so that the weak CTL levels become increasingly more dominant relative to antibodies. This shift results in onset of liver pathology as the virus evolves towards increased levels of antigenic escape. Therefore, the relative balance of the immune response can be a decisive factor that determines whether patients are asymptomatic or whether pathology is observed. Virus evolution can shift this balance towards pathology over time. Theoretical results are discussed in the context of published data.

Cancer vaccines: progress reveals new complexities.

A decade ago, it seemed clear that our burgeoning knowledge of the molecular identities of tumor-associated antigens and a deeper understanding of basic immunology would point the way to an effective therapeutic cancer vaccine. Significant progress has been made and objective regressions after immune-based treatments are observed in some patients — even in those with bulky, metastatic disease. Notwithstanding this progress, we do not yet have a cancer vaccine in hand that can reliably increase patient survival or induce tumor destruction. The creation of therapeutic cancer vaccines has proven to be an enormous challenge, and many of the strategies learned in the development of highly successful vaccines against infectious agents simply do not apply to cancer vaccines. Issues of antigenic change and immune escape are present in both antitumor and antiviral situations. However, one big difference between antiviral and antitumor vaccines is that the former are preventative whereas the latter are generally expected to be therapeutic. Another problem with the targeting of tumor antigens relates to their poor immunogenicity. Tumor antigens appear to be relatively well tolerated in the host, perhaps because many of these antigens are also expressed in normal tissues. In this context, a successful cancer vaccine raises the specter of autoimmune attack if the vaccines are ultimately powerful enough to eliminate cancer cells. In this review, we highlight new challenges that have been revealed by recent progress in the field of tumor immunology. We then attempt to outline a future plan for cancer immunotherapy.

Viral persistence: HIV's strategies of immune system evasion.

In contrast to most animal viruses, infection with the human and simian immunodeficiency viruses results in prolonged, continuous viral replication in the infected host. Remarkably, viral persistence is not thwarted by the presence of apparently vigorous, virus-specific immune responses. Several factors are thought to contribute to persistent viral replication, most notably the destruction of virus-specific T helper cells, the emergence of antigenic escape variants, and the expression of an envelope complex that structurally minimizes antibody access to conserved epitopes. Not as well understood, though potentially important, is the ability of at least one viral encoded protein (Nef) to prevent presentation of viral antigens in the context of major histocompatibility complex. The future success of antiviral therapies and vaccination strategies may depend largely on understanding how and to what degree each of these factors (and presumably others) contributes to immune evasion.

CD8 memory, immunodominance, and antigenic escape.

Previous theoretical work has suggested that efficient virus control or clearance requires antigen-independent persistence of memory cytotoxic T lymphocyte precursors (CTLp), and that failure to generate such memory CTLp can result in persistent infection and eventual loss of virus control. Here we use mathematical models to investigate the relationship between virus control, the clonal composition of the CTL response and the chance of the virus to evolve antigenic escape. In the presence of efficient memory CTLp, virus is controlled at very low levels by a broad CTL response directed against multiple epitopes. In this case, antigenic escape of the virus population is expected to take a very long time. On the other hand, if the CTL response is short lived in the absence of antigen, virus replicates at higher levels and is only opposed by a narrow CTL response, characterized by an immunodominant CTL clone. In this case, antigenic escape is expected to evolve in a short period of time, resulting in progressive loss of virus control. We discuss our findings in relation to data from HIV-1-infected patients.

Cancer-induced Defective Cytotoxic T Lymphocyte Effector Function: Another Mechanism How Antigenic Tumors Escape Immune-mediated Killing

The notion that a deficit in immune cell functions permits tumor growth has received experimental support with the discovery of several different biochemical defects in T lymphocytes that infiltrate cancers. Decreased levels of enzymes involved with T-cell signal transduction have been reported by several laboratories, suggesting that tumors or host cells recruited to the tumor site actively down-regulate antitumor T-cell immune response. This permits tumor escape from immune-mediated killing. The possibility that defects in T-cell signal transduction can be reversed, which would potentially permit successful vaccination or adoptive immunotherapy, motivates renewed interest in the field. Summarizing the literature concerning tumor-induced T-cell dysfunction, we focus on the end stage of immune response to human cancer, that of defective cytotoxic T lymphocyte killing function. Based on the data from several laboratories, we hypothesize a biochemical mechanism that accounts for the unusual phenotype of antitumor T-cell accumulation in tumors, but with defective killing function.

Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.

Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.

Identification of antigenic escape variants in an immunodominant epitope of hepatitis C virus.

Numerous investigators have postulated that one mechanism by which hepatitis C virus (HCV) may evade the immune system is through the formation of escape mutants. This hypothesis is based largely on the observed mutability of the viral genome resulting in evolution of diverse quasispecies over the course of infection. That such diversification is a product of viral RNA polymerase infidelity, immune-driven selection or a combination of the two processes has not been addressed. We have examined sequence variability in a specific segment of HCV RNA encoding a known immunodominant region of the viral helicase, amino acids 358-375 of the non-structural 3 protein. Using sequence-specific oligonucleotide probe hybridization and automated DNA sequencing, we report a high frequency of mutations, essentially all of which result in amino acid replacements. To assess the biological impact of such mutations, corresponding chemically synthesized peptides were compared to wild-type peptide in T cell proliferation assays. We observed that a sizeable fraction of such peptides stimulated attenuated or negligible levels of proliferation by peripheral T cells from a chronically infected patient. This observation is consistent with expectations for immune-mediated selection of escape variants at the epitope level. We postulate that such a mechanism may be important in the immunopathogenesis of HCV infections.

Blocking antibodies inhibit complete African swine fever virus neutralization

A persistent non-neutralized African swine fever virus (ASFV) fraction is found with most convalescent swine sera in in vitro neutralization assays. To study this phenomenon, antisera from convalescent pigs infected with different virus isolates and showing complete or incomplete virus neutralization were used. Different experiments determined that incomplete neutralization of ASFV is caused neither by virus aggregation, nor low affinity or stability of virus-antibody complexes. Additionally, attempts to purify antigenic escape mutant viruses from the persistent fraction was also unsuccessful. Nevertheless, competition experiments between sera demonstrated that antibodies present in sera showing persistent fraction inhibited the complete neutralization mediated by antibodies present in sera which neutralize 100% of virus infectivity. These results suggest that induction of blocking antibodies during ASFV infection could represent the main cause for the persistent surviving virus fraction observed in neutralization assays and could also explain the persistent infections observed in some convalescent pigs.

Immune responses against multiple epitopes

The current understanding of antigenic escape dynamics is based on models with single epitopes. The usual idea is that a mutation which enables a pathogen (virus, bacteria, etc) to escape from a given immune response confers a selective advantage. The “escape mutant” may then increase in abundance until it induces a new specific response against itself. In this paper a new picture is developed, based on mathematical models of immune responses against several epitopes; the simplest such models can have very complicated dynamics, with some surprising features. The emergence of an escape mutant can shift the immunodominant response to another epitope. Even in the absence of mutations, antigenic oscillation is found, with distinct peaks of different virus variants and fluctuations in the size and specificity of the immune responses. The model also provides a general theory for immunodominance in the presence of antigenic variation. Immunodominance is determined by the immunogenicity and by the antigenic diversity of the competing epitopes. Antigenic oscillations and fluctuations in the cytotoxic T-lymphocyte response have been observed in infections with the human immunodeficiency virus (HIV). Shifting the immune responses to weaker epitopes can represent a mechanism for disease progression based on evolutionary dynamics and antigenic diversity of the virus.

Antigenic oscillations and shifting immunodominance in HIV-1 infections.

A typical protein antigen contains several epitopes that can be recognized by cytotoxic T lymphocytes (CTL), but in a characteristic antiviral immune response in vivo, CTL recognize only a small number of these potential epitopes, sometimes only one, this phenomenon is known as immunodominance. Antigenic variation within CTL epitopes has been demonstrated for the human immunodeficiency virus HIV-1 (ref. 11) and other viruses and such 'antigenic escape' may be responsible for viral persistence. Here we develop a new mathematical model that deals with the interaction between CTL and multiple epitopes of a genetically variable pathogen, and show that the nonlinear competition among CTL responses against different epitopes can explain immunodominance. This model suggests that an antigenically homogeneous pathogen population tends to induce a dominant response against a single epitope, whereas a heterogeneous pathogen population can stimulate complicated fluctuating responses against multiple epitopes. Antigenic variation in the immunodominant epitope can shift responses to weaker epitopes and thereby reduce immunological control of the pathogen population. These ideas are consistent with detailed longitudinal studies of CTL responses in HIV-1 infected patients. For vaccine design, the model suggests that the major response should be directed against conserved epitopes even if they are subdominant.