Monoclonal Antibody-resistant Mutants Research Articles

Selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants

The use of monoclonal neutralizing antibodies (mNAbs) is being actively pursued as a viable intervention for the treatment of Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2) infection and associated coronavirus disease 2019 (COVID-19). While highly potent mNAbs have great therapeutic potential, the ability of the virus to mutate and escape recognition and neutralization of mNAbs represents a potential problem in their use for the therapeutic management of SARS-CoV-2. Studies investigating natural or mNAb-induced antigenic variability in the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) glycoprotein, and their effects on viral fitness are still rudimentary. In this manuscript we described experimental approaches for the selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants (MARMs) in cultured cells. The ability to study SARS-CoV-2 antigenic drift under selective immune pressure by mNAbs is important for the optimal implementation of mNAbs for the therapeutic management of COVID-19. This will help to identify essential amino acid residues in the viral S glycoprotein required for mNAb-mediated inhibition of viral infection, to predict potential natural drift variants that could emerge upon implementation of therapeutic mNAbs, as well as vaccine prophylactic treatments for SARS-CoV-2 infection. Additionally, it will also enable the assessment of MARM viral fitness and its potential to induce severe infection and associated COVID-19 disease.

Monoclonal antibody resistant mutant of Peste des petits ruminants vaccine virus.

The available vaccines for control of Peste des petits ruminants do not favour differentiation of infected and vaccinated animals (DIVA). Hence, the present study was aimed to isolate and characterize monoclonal antibody resistant mutant of an Indian strain of vaccine virus "PPRV-Sungri/96" under selection pressure of virus neutralizing monoclonal antibody '4B11' specific to haemagglutinin (H) protein. We successfully isolated five monoclonal antibody resistant (mAr) mutants (PPRV-RM5, PPRV-RM6, PPRV-RM7, PPRV- E6 and PPRV- E7). The mAr mutants did not react with the anti-H mAb 4B11 whereas reacted with control anti-nucleoprotein mAb 4G6, similar to the parent vaccine virus "PPRV-Sungri/96" in indirect ELISA, cell ELISA and indirect immunofluorescence test. Cytometry analysis of mAr mutants revealed loss of binding to mAb 4B11 while maintaining binding to mAb 4G6, more or less similar to "PPRV-Sungri/96". The sequence analysis of the H-protein gene of the mAr mutants resulted in identification of two nucleotide changes leading to amino acid substitutions at position 263 and 502 (L263P and R502P) of the H protein indicating that the epitope of mAb 4B11 could be conformational in nature. Though, mAr mutant grew to a similar titre as parent vaccine virus (PPRV-Sungri/96), the in vivo work in goats to study the mAr mutant as possible negative marker vaccine candidate could not be successfully proved with mAb 4B11 based competitive ELISA. However, one of the nucleotide change (T-C) at position 788, unique to mAr mutant virus resulted in abolition of a restriction enzyme recognition site (BglII). This could be used to differentiate mAr mutant vaccine virus from other available vaccine and field strains using restriction fragment length polymorphism. However, the mAr mutant PPRV-E6 cannot be used as a candidate strain for DIVA vaccine as immune response against it cannot be differentiated based on serology.

Evolution of serotype A foot-and-mouth disease virus capsid under neutralizing antibody pressure in vitro

In this study, the Indian foot-and-mouth disease virus (FMDV) vaccine strain (A IND 40/2000) was passaged under homologous bovine convalescent serum (BCS) pressure to gain insight into the evolutionary dynamics of the antigenic sites. A considerable drop in the neutralization titres of the BCS for the isolated variants as compared to the parental population in either virus neutralization or plaque reduction neutralization test was observed. T143K substitution preceding the integrin binding ‘RGD’-motif in the βG-βH loop of VP1 was found to be selected consistently and exclusively under immune pressure. By virtue of its location within an immunodominant site, sequence heterogeneity observed in the field viruses and residues already mapped in the neutralizing monoclonal antibody resistant mutants, position 143 in VP1 was predicted to be a critical residue of an important neutralizing epitope in serotype A FMDV. Using next-generation sequencing approach, the gradual overtaking of the originally dominating major variant by a minor one under a selective environment could be demonstrated. In the control passage regimen, VP2 E131K substitution was fixed within the heparan sulfate binding pocket probably as a result of adaptation to use alternative cellular receptors. But at the same time, these substitutions arising under selection forces other than immune pressure changed the antigenic behaviour of the virus inadvertently.

A Single Mutation in the Glycophorin A Binding Site of Hepatitis A Virus Enhances Virus Clearance from the Blood and Results in a Lower Fitness Variant

Hepatitis A virus (HAV) has previously been reported to bind to human red blood cells through interaction with glycophorin A. Residue K221 of VP1 and the surrounding VP3 residues are involved in such an interaction. This capsid region is specifically recognized by the monoclonal antibody H7C27. A monoclonal antibody-resistant mutant with the mutation G1217D has been isolated. In the present study, the G1217D mutant was characterized physically and biologically in comparison with the parental HM175 43c strain. The G1217D mutant is more sensitive to acid pH and binds more efficiently to human and rat erythrocytes than the parental 43c strain. In a rat model, it is eliminated from serum more rapidly and consequently reaches the liver with a certain delay compared to the parental 43c strain. In competition experiments performed in vivo in the rat model, the G1217D mutant was efficiently outcompeted by the parental 43c strain. Only in the presence of antibodies reacting specifically with the parental 43c strain could the G1217D mutant outcompete the parental 43c strain in serum, although the latter still showed a remarkable ability to reach the liver. Altogether, these results indicate that the G1217D mutation induces a low fitness phenotype which could explain the lack of natural antigenic variants of the glycophorin A binding site.

Density-dependent selection in vesicular stomatitis virus.

We used vesicular stomatitis virus to test the effect of complementation on the relative fitness of a deleterious mutant, monoclonal antibody-resistant mutant (MARM) N, in competition with its wild-type ancestor. We carried out competitions of MARM N and wild-type populations at different multiplicities of infection (MOIs) and initial ratios of the wild type to the mutant and found that the fitness of MARM N relative to that of the wild type is very sensitive to changes in the MOI (i.e., the degree of complementation) but depends little, if at all, on the initial frequencies of MARM N and the wild type. Further, we developed a mathematical model under the assumption that during coinfection both viruses contribute to a common pool of protein products in the infected cell and that they both exploit this common pool equally. Under such conditions, the fitness of all virions that coinfect a cell is the average fitness in the absence of coinfection of that group of virions. In the absence of coinfection, complementation cannot take place and the relative fitness of each competitor is only determined by the selective value of its own products. We found good agreement between our experimental results and the model predictions, which suggests that the wild type and MARM N freely share all of their gene products under coinfection.

Immune Recognition of Swine Vesicular Disease Virus Structural Proteins: Novel Antigenic Regions That Are Not Exposed in the Capsid

Swine vesicular disease virus (SVDV) is an enterovirus of the Picornaviridae family that belongs to the coxsackievirus B group. A number of antigenic sites have been identified in SVDV by analysis of neutralizing monoclonal antibody-resistant mutants and shown to be exposed on the surface of the capsid. In this paper we have identified seven new immunodominant antigenic regions in SVDV capsid proteins by a peptide scanning method, using a panel of sera from infected pigs. When these antigenic regions were located in the capsid by using a computer-generated three-dimensional model of the virion, one was readily exposed on the surface of the virus and the remaining sites were located facing the inner side of the capsid shell, at subunit contacts, or in the interior of the subunit structure.

An estimation of the nucleotide substitution rate at defined positions in the influenza virus haemagglutinin gene.

The mutation rates to a viable mar (monoclonal antibody-resistant mutant) genotype of wild-type influenza (A/Victoria/3/75; H3N2) virus or its mutator variant strains have been previously determined. In order to estimate the mutation rates per nucleotide position, the sequence alterations present in 44 mar mutants isolated from either the wild-type or the mut43 mutator strain have been determined. These mar mutants were selected with either of two non-overlapping, haemagglutinin-specific, monoclonal antibodies (2G10 and p7). Most of the protein changes were identified as substitutions of large, charged amino acids for glycine residues, as the result of G to A transitions. Particularly interesting amino acid changes, not previously reported, were observed in the p7 monoclonal antibody-specific mutants, in which only Gly to Ser and Gly to Asp at position 226 were detected. The identification of the nucleotide substitutions responsible for the mar phenotype has allowed the calculation of approximate values for the total mutation rates at these positions.

Multiple amino acid substitutions in the HN protein of the paramyxovirus, SV5, are selected for in monoclonal antibody resistant mutants.

Monoclonal antibody resistant (MAR) mutants (which escaped antibody-mediated neutralization) were selected from simian (W 3) and human (LN) isolates of simian virus 5 (SV 5), using monoclonal antibodies (MAbs) specific for antigenic sites 4 and 5 on the HN glycoprotein. Resistance correlated with an inability of the selecting antibody to bind with the respective MAR mutants. Sequence comparisons between parental and mutant HN proteins revealed multiple non-adjacent amino acid substitutions in the majority of MAR mutants. The same multiple substitutions were identified in mutants selected from both the LN and W 3 isolates of SV 5. Furthermore, different mutations on the primary sequence of the HN protein conferred resistance to the same MAb.

Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses

We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.

Sequence analysis of monoclonal antibody resistant mutants of type O foot and mouth disease virus: Evidence for the involvement of the three surface exposed capsid proteins in four antigenic sites

Sequence analysis of monoclonal antibody resistant mutants of type O foot and mouth disease virus has been performed. Distinct clusters of amino acid substitutions conferring resistance to neutralization at each of the four previously defined antigenic sites (McCahon et al., 1989, J. Gen. Virol. 70, 639–645) have been identified. One site corresponds to the well-known 140–160 region of VP1, a second site is also on VP1, one site is on VP2, and the fourth site is on VP3. All of the amino acid substitutions identified are located on the surface of the virus. Despite the differences in three-dimensional structure between FMDV and other picornaviruses the neutralizing antigenic sites occur in analogous positions on the capsid surface.

Localization on the herpes simplex virus type 1 genome of a region encoding proteins involved in adsorption to the cellular receptor

We have previously shown that aminoglycosides such as neomycin and the polyamino acids polylysine and polyarginine selectively inhibit the binding of herpes simplex virus type 1 (HSV-1) to the cellular receptor, whereas HSV-2 infection is unaffected. In the present study we took advantage of this difference between HSV-1 and HSV-2 by using HSV(-1)-HSV(-2) intertypic recombinants to locate a region on the HSV-1 genome encoding proteins affecting the binding of the virion to the cellular receptor. The results were consistent with those obtained by marker rescue experiments. The identified region, which mapped between coordinates 0.580 and 0.687, contains two partial and eight complete genes, including the glycoprotein C (gC) gene and two others with potential transmembrane sequences. Various gC monoclonal antibody-resistant mutants of HSV-1 as well as a mutant completely lacking gC were found to be fully sensitive to neomycin, suggesting that gC is not the site of drug sensitivity and is not essential for adsorption of virus to the cellular receptor. However, the rate of adsorption was reduced in the absence of gC, indicating a facilitating function of the glycoprotein. The universal nature of this HSV-1 receptor binding was revealed by the similarity in drug sensitivity of infectivity in four different cell lines from various tissues and species.

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.

Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions

Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach.

Evidence for At Least Four Antigenic Sites on Type O Foot-and-Mouth Disease Virus Involved in Neutralization; Identification by Single and Multiple Site Monoclonal Antibody-resistant Mutants

Neutralizing monoclonal antibodies raised against type O foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type O convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed.

Antigenic analysis of a major neutralization site of herpes simplex virus glycoprotein D, using deletion mutants and monoclonal antibody-resistant mutants.

Herpes simplex virus glycoprotein D is a component of the virion envelope and appears to be involved in attachment, penetration, and cell fusion. Monoclonal antibodies against this protein can be arranged in groups on the basis of a number of biological and biochemical properties. Group I antibodies are type common, have high complement-independent neutralization titers, and recognize discontinuous (conformational) epitopes; they are currently being used in several laboratories to study the functions of glycoprotein D. We have used a panel of neutralization-resistant mutants to examine the relationships between these antibodies in detail. We found that they can be divided into two subgroups, Ia and Ib, such that mutations selected with Ia antibodies have little or no effect on binding and neutralization by Ib antibodies and vice versa. In addition, Ia antibodies are able to bind deletion and truncation mutants of glycoprotein D that Ib antibodies do not recognize, suggesting that their epitopes are physically distinct. However, with one exception, Ia and Ib antibodies block each other strongly in binding assays with purified glycoprotein D, whereas antibodies from other groups have no effect. We have therefore defined the sum of the Ia and Ib epitopes as antigenic site 1.

Antigen chimaeras of poliovirus as potential new vaccines

Polioviruses occur as three distinct serotypes, 1, 2 and 3, and are composed of a single-stranded positive-sense RNA genome of approximately 7,450 nucleotides enclosed in an icosahedral particle of diameter 27 nm. The three-dimensional crystallographic structure of poliovirus type 1 has been determined at 2.9 A resolution, providing a detailed knowledge of the folding and arrangement of the individual virus proteins, VP1-VP4. From this and the characterization of monoclonal antibody-resistant mutants, the amino acids contributing to antigenic sites have been identified and located on the surface of the virus particle. Here we describe the construction and characterization of a poliovirus chimaera having a defined region of type 3 inserted into type 1. This virus has composite antigenicity and the substitute site is immunogenic in small animals and primates. The ability to construct such viruses has implications for the design of improved poliovirus vaccine strains or vaccines against other picornaviruses, such as hepatitis A.

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.

Isolation and characterization of monoclonal antibody-resistant mutants of Newcastle disease virus.

Neutralizing monoclonal antibodies incorporated into plaque assay overlay medium were used to select antibody-resistant (AbR) mutants of both the Herts (using antifusion protein monoclonal 481) and Beaudette C (using anti-haemagglutinin-neuraminidase protein monoclonal 445) strains of Newcastle disease virus at the permissive temperature of 34 degrees C. Certain of the Herts, but none of the Beaudette C, AbR mutants were also temperature-sensitive (ts-) and failed to form plaques at the non-permissive temperature of 41.5 degrees C. [35S]Methionine-labelled proteins from chick embryo fibroblasts infected with wild-type, ts+ AbR and ts- AbR virus when separated by two-dimensional polyacrylamide gel electrophoresis revealed a variety of changes in the isoelectric point of the fusion protein F (using monoclonal 481) and the haemagglutinin-neuraminidase protein HN (using monoclonal 445). The ts+ 'revertants' of ts- AbR mutants remained AbR and also showed changed isoelectric points in the F protein.