Rapid selection of genetic and antigenic variants of foot-and-mouth disease virus during persistence in cattle.

Rapid evolution of foot-and-mouth disease virus (FMDV) is documented during persistent infections of cattle. The carrier state was established experimentally with plaque-purified FMDV of serotype C3. Virus was recovered from the esophageal pharyngeal area of the animals up to 539 days postinfection. Analysis of capsid proteins by electrofocusing and by electrophoretic mobility of the genomic poly(C)-rich tract suggested heterogeneity in several isolates and sequential dominance of viral subpopulations. Nucleotide sequences of the VP1-coding region of the parental FMDV C3 clones and of seven isolates from the carrier cattle showed point mutations that represented rates of fixation of mutations of 0.9 X 10(-2) to 7.4 X 10(-2) substitutions per nucleotide per year; 59% of the base changes led to amino acid substitutions, some of which were located within residues 135 to 151, a region involved in neutralization of FMDV. In the esophageal pharyngeal fluid samples, FMDV C3-neutralizing activity was present. Antigenic variation was demonstrated with monoclonal antibodies raised against FMDV C3. Two isolates from carrier cattle differed from the parental virus by 10(2)- or 10(3)-fold decreased reactivity with neutralizing monoclonal antibodies. We suggest that persistent, inapparent infections of ruminants, in addition to being a reservoir of virus, may promote the rapid selection of antigenically variant FMDVs.     

Column selection of antigenic variants from tumors. YAC (Moloney) lymphoma variants with reduced antigen expression

Serial pretreatment of YAC lymphoma cells with anti H-2^a serum and anti-Ig column selection to remove cells with high surfaceH-2 concentration, followed by intermittent transfer into mice has led to the establishment of two variant sublines with low H-2^a antigen expression and only a slightly reduced Moloney virus-determined cell surface antigen (MCSA) expression compared to the original YAC line. Both sublines had an increased number of chomosomes with a modal number of 62 and 57, as compared to 39 in YAC. In contrast to YAC, the variants were freely homotransplantable across theH-2 barrier. They also resisted the rejection response of YAC-preimmunized semisyngeneic mice. Parallel selection against MCSA resulted in a variant with reduced MCSA, but unchanged H-2^a expression and karyotype. This subline failed to grow across the allograft barrier, and showed only a slightly increased ability to grow in preimmunized semisyngeneic mice.These results suggest thatH-2 expression may play an important role in the ability of antigenic tumor cells to be rejected in specifically preimmunized, genetically compatible recipients.     

DNA polymerases during tumor growth

DNA polymerase activity was studied as a function of stage of tumor growth and correlated with DNA synthesis measured by 3H-TdR uptake. Considerable variations in DNA synthesis activity occur at different growth stages and following host death. DNA alpha-polymerase activity did vary with growth stage in the ascites tumor. However, it did not have a clear correlation with DNA synthesis or with tumor growth. No striking fall in DNA polymerase enzyme levels occurred as the ascites tumor reached stationary phase in contrast to reports in some cell culture systems. A decrease occurred with advanced tumor stage and after host death. DNA beta-polymerase activity did not change with tumor growth stage.     

Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection.

Antigenic variants of foot-and-mouth disease virus (FMDV) were generated and frequently became dominant in clonal populations of FMDV (clone C-S8c1) grown in the absence of anti-FMDV antibodies. We have now passaged eight samples of the same FMDV clone in the presence of a limited amount of neutralizing polyclonal antibodies directed to the major antigenic site A of capsid protein VP1. Complex populations of variants showing increased resistance to polyclonal sera and to site A-specific monoclonal antibodies were selected. Some populations exhibited marked decreases in viral fitness. Multiple amino acid replacements within site A--and also elsewhere in VP1--accumulated upon passage of the virus in either the absence or the presence of neutralizing antibodies. However, antigenically critical replacements at one position in site A occurred repeatedly in FMDV passaged under antibody selection, but they were never observed in many passages carried out either in the absence of antiviral antibodies or in the presence of an irrelevant antiviral serum. Thus, even though antigenic variation of FMDV can occur in the absence or presence of immune selection, critical replacements which lead to important changes in antigenic specificity were observed only as a result of selection by neutralizing antibodies.     

A protocol for detection of H-Y antigenic variants

A skin grafting protocol is described for finding H-Y antigenic variants. The method is applicable regardless of the location of the structural gene(s) for this antigen (X, Y, or autosomal). Use of this protocol revealed no evidence for H-Y antigenic variation between C57BL/6J and strains 129/J, A.BY/SnJ, C3H.SW/SnJ, and LP/J.     

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Comparative immunologic effectiveness of variants of the DTP vaccine

The immunological effectiveness of two batches of adsorbed DPT vaccine, the batch with the normal content of antigens (control) and the batch with the content of diphtheria and tetanus toxoids reduced to 20 Lf/ml and 5 BU/ml respectively (test batch), has been studied under the conditions of controlled trial. As a result, the reduction of the antigenic content of adsorbed DPT vaccine has been found to exert no negative influence on the immunological effectiveness of the diphtheria, tetanus, and pertussis components of this preparation under the conditions of the new immunization schedule.     

Influence of the mutant spectrum in viral evolution: focused selection of antigenic variants in a reconstructed viral quasispecies

RNA viruses replicate as complex mutant distributions termed viral quasispecies. Despite this, studies on virus populations subjected to positive selection have generally been performed and analyzed as if the viral population consisted of a defined genomic nucleotide sequence; such a simplification may not reflect accurately the molecular events underlying the selection process. In the present study, we have reconstructed a foot-and-mouth disease virus quasispecies with multiple, low-frequency, genetically distinguishable mutants that can escape neutralization by a monoclonal antibody. Some of the mutants included an amino acid substitution that affected an integrin recognition motif that overlaps with the antibody-binding site, whereas other mutants included an amino acid substitution that affected antibody binding but not integrin recognition. We have monitored consensus and clonal nucleotide sequences of populations passaged either in the absence or the presence of the neutralizing antibody. In both cases, the populations focused toward a specific mutant that was surrounded by a cloud of mutants with different antigenic and cell recognition specificities. In the absence of antibody selection, an antigenic variant that maintained integrin recognition became dominant, but the mutant cloud included as one of its minority components a variant with altered integrin recognition. Conversely, in the presence of antibody selection, a variant with altered integrin recognition motif became dominant, but it was surrounded by a cloud of antigenic variants that maintained integrin recognition. The results have documented that a mutant spectrum can exert an influence on a viral population subjected to a sustained positive selection pressure and have unveiled a mechanism of antigenic flexibility in viral populations, consisting in the presence in the selected quasispecies of mutants with different antigenic and cell recognition specificities.     

The rate of in vivo selection of highly metastatic and resistance-inhibiting variants of tumor cells

The rate of in vivo selection of tumour cells of STHE strain (spontaneously transformed in vitro hamster embryo cells, previously not selected in vivo) after their intravenous inoculation to normal Syrian hamsters was studied. At different time intervals after inoculation tumour cells were isolated from the lung tissue of individual animals, cultured in vitro and the their metastatic activity (MA) and natural resistance-depressing activity (RDA) were studied. It was demonstrated that selection of highly metastatic variants of tumour cells begins immediately after inoculation; on days 5-6 after inoculation the presence of such variants is observed in the majority of animals. Selection of resistance-depressing variants of tumour cells (determined in a less sensitive test) was first noticed on days 9-10 after inoculation of tumour cells. The correlation between MA and RDA of tumour cells was observed. The data obtained give evidence in favour of preexistence of genetic MA and RDA variants of tumour cells in the original population of tumour cells.     

Nonspecific antigenic suppression of the formation of immunologic memory to heteroerythrocytes

Spleen cells (SC) of mice immunized with sheep red blood cells contain factor that suppresses primary immune response and the formation of immunologic memory. During studies of antigen-specific suppression of the immunogenesis, it has been discovered that suppressor factor of SC non-specifically blocks the development of immunologic memory for the other antigen (rat red cells) without affecting primary immune response to this antigen administration. It is assumed that at an early stage the cells responsible for the formation of immunologic memory are more sensitive to the non-specific effect of suppressor factor than those involved in the generation of primary immune response.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection.

Previous results from our laboratory have demonstrated that equine infectious anemia virus displays structural variations in its surface glycoproteins and RNA genome during passage and chronic infections in experimentally infected Shetland ponies (Montelaro et al., J. Biol. Chem. 259:10539-10544, 1984; Payne et al., J. Gen. Virol. 65:1395-1399, 1984). The present study was undertaken to obtain an antigenic and biochemical characterization of equine infectious anemia virus isolates recovered from an experimentally infected pony during sequential disease episodes, each separated by intervals of only 4 to 8 weeks. The virus isolates could be distinguished antigenically by neutralization assays with serum from the infected pony and by Western blot analysis with a monoclonal antibody against the major surface glycoprotein gp90, thus demonstrating that novel antigenic variants of equine infectious anemia virus predominate during each clinical episode. The respective virion glycoproteins displayed different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels, indicating structural variation. Tryptic peptide and glycopeptide maps of the viral proteins of each virus isolate revealed biochemical alterations involving amino acid sequence and glycosylation patterns in the virion surface glycoproteins gp90 and gp45. In contrast, no structural variation was observed in the internal viral proteins pp15, p26, and p9 from any of the four virus isolates. Oligonucleotide mapping experiments revealed similar but unique RNase T1-resistant oligonucleotide fingerprints of the RNA genomes of each of the virus isolates. Localization of altered oligonucleotides for one virus isolate placed two of three unique oligonucleotides within the predicted env gene region of the genome, perhaps correlating with the structural variation observed in the envelope glycoproteins. Thus these results support the concept that equine infectious anemia virus is indeed capable of relatively rapid genomic variations during replication, some of which result in altered glycoprotein structures and antigenic variants which are responsible for the unique periodic disease nature observed in persistently infected animals. The findings of envelope specific differences in isolates of visna virus and of human T-cell lymphotropic virus III (acquired immune deficiency syndrome-related virus) suggest that this variation may be a common characteristic of the subfamily Lentivirinae.     

Organ-specific human prostate beta-globulin during tumor growth

An organ specific protein antigen having an electrophoretic mobility of beta-globulin was demonstrated in human prostate. Its physicochemical properties (relative electrophoretic mobility, diffusion coefficient, molecular weight and relation to different precipitating agents) were determined. The indirect immunofluorescence technique revealed that this antigen is synthesized by the glandular epithelium of the main glands. A statistically significant decrease was found in the concentration of prostatic beta-globulin in tumors of the prostate. The synthesis of this protein was shown to start only in the period of puberty.     

Bias of selection on human copy-number variants

Although large-scale copy-number variation is an important contributor to conspecific genomic diversity, whether these variants frequently contribute to human phenotype differences remains unknown. If they have few functional consequences, then copy-number variants (CNVs) might be expected both to be distributed uniformly throughout the human genome and to encode genes that are characteristic of the genome as a whole. We find that human CNVs are significantly overrepresented close to telomeres and centromeres and in simple tandem repeat sequences. Additionally, human CNVs were observed to be unusually enriched in those protein-coding genes that have experienced significantly elevated synonymous and nonsynonymous nucleotide substitution rates, estimated between single human and mouse orthologues. CNV genes encode disproportionately large numbers of secreted, olfactory, and immunity proteins, although they contain fewer than expected genes associated with Mendelian disease. Despite mouse CNVs also exhibiting a significant elevation in synonymous substitution rates, in most other respects they do not differ significantly from the genomic background. Nevertheless, they encode proteins that are depleted in olfactory function, and they exhibit significantly decreased amino acid sequence divergence. Natural selection appears to have acted discriminately among human CNV genes. The significant overabundance, within human CNVs, of genes associated with olfaction, immunity, protein secretion, and elevated coding sequence divergence, indicates that a subset may have been retained in the human population due to the adaptive benefit of increased gene dosage. By contrast, the functional characteristics of mouse CNVs either suggest that advantageous gene copies have been depleted during recent selective breeding of laboratory mouse strains or suggest that they were preferentially fixed as a consequence of the larger effective population size of wild mice. It thus appears that CNV differences among mouse strains do not provide an appropriate model for large-scale sequence variations in the human population.     

The selection of tumor variants with altered expression of classical and nonclassical MHC class I molecules: implications for tumor immune escape

Tumor immune escape variants can be identified in human and experimental tumors. A variety of different strategies are used by tumor cells to avoid recognition by different immune effector mechanisms. Among these escape routes, alteration of MHC class I cell surface expression is one of the mechanisms most widely used by tumor cells. In this review we focus our attention on the T-cell immune selection of MHC class I–deficient tumor variants. Different altered MHC class I phenotypes that originate from multiple molecular mechanisms can be identified in human tumors. MHC-deficient tumor clones can escape T-cell immune responses, but are in theory more susceptible to NK-cell–mediated lysis. In this context, we also review the controversial issue of the aberrant expression of nonclassical HLA class I molecules, particularly HLA-G, in tumors. This expression may be relevant in tumor cells that have lost the capacity to interact with NK inhibitory receptors—namely, those tumor cells with no HLA-B or HLA-C expression. Most published studies have not analyzed these possibilities and do not provide information about the complete HLA-A, HLA-B, or HLA-C molecule profiles of the tumors studied. In contrast, HLA-E has been reported to be expressed in some tumor cell lines with very low HLA-A, HLA-B, and HLA-C expression, suggesting that HLA-E may indeed, in some cases, play a role by inhibiting NK lysis of cells that otherwise would be destroyed by NK cells. Finally, we provide evidence that the status of the immune system in the tumor-bearing animal is capable of defining the MHC profile of the tumor cells. In other words, MHC class I–negative metastatic colonies are produced in immunocompetent animals, and MHC class I–positive colonies in T-cell immunodeficient individuals.This article forms part of the Symposium in Writing Tumor escape from the immune response, published in Vol. 53.     

Receptor-binding characteristics of monoclonal antibody-selected antigenic variants of influenza virus.

Erythrocytes modified to different extents with periodate were used in hemagglutination assays to investigate the binding properties of antihemagglutinin monoclonal antibody-selected antigenic variants of X-31 influenza virus. The results allowed differentiation of groups of variants and are discussed in relation to the nature of the amino acid substitutions in the variant hemagglutinins and their molecular locations relative to the receptor-binding site.