Linear B-cell Epitope Mapping Research Articles

A novel precision-serology assay for SARS-CoV-2 infection based on linear B-cell epitopes of Spike protein.

The COVID-19 pandemic illustrates the need for serology diagnostics with improved accuracy. While conventional serology based on recognition of entire proteins or subunits thereof has made significant contribution to the antibody assessment space, it often suffers from sub-optimal specificity. Epitope-based, high-precision, serology assays hold potential to capture the high specificity and diversity of the immune system, hence circumventing the cross-reactivity with closely related microbial antigens. We herein report mapping of linear IgG and IgA antibody epitopes of the SARS-CoV-2 Spike (S) protein in samples from SARS-CoV-2 exposed individuals along with certified SARS-CoV-2 verification plasma samples using peptide arrays. We identified 21 distinct linear epitopes. Importantly, we showed that pre-pandemic serum samples contain IgG antibodies reacting to the majority of protein S epitopes, most likely as a result of prior infection with seasonal coronaviruses. Only 4 of the identified SARS-CoV-2 protein S linear epitopes were specific for SARS-CoV-2 infection. These epitopes are located at positions 278-298 and 550-586, just proximal and distal to the RBD, as well as at position 1134-1156 in the HR2 subdomain and at 1248-1271 in the C-terminal subdomain of protein S. To substantiate the applicability of our findings, we tested three of the high-accuracy protein S epitopes in a Luminex assay, using a certified validation plasma sample set from SARS-CoV-2 infected individuals. The Luminex results were well aligned with the peptide array results, and correlated very well with in-house and commercial immune assays for RBD, S1 and S1/S2 domains of protein S. We present a comprehensive mapping of linear B-cell epitopes of SARS-CoV-2 protein S, that identifies peptides suitable for a precision serology assay devoid of cross-reactivity. These results have implications for development of highly specific serology test for exposure to SARS-CoV-2 and other members of the coronaviridae family, as well as for rapid development of serology tests for future emerging pandemic threats.

Viroinformatics study: polytope mapping of envelope glycoprotein to tackle HIV-2 infection and develop vaccine candidate

Human Immunodeficiency Virus type 2 (HIV-2) has been identified to exhibit an ability to resist antiretroviral administration and many scientists has predicted increases in the pathogenicity of HIV-2. The development of a vaccine against the type 1 virus (HIV-1) infection has reached the phase 3 clinical trial stage, but currently there is no information on the development of a vaccine against HIV-2. Vaccine development to trigger an increase in the coverage of the expansion of protection can be done through B cell polytope. This study aims to provide an important preliminary for the construction of vaccine candidates by identifying the peptides that make up the B cell polytope in the HIV-2 envelope glycoprotein region. The HIV-2 sequence was obtained from the database. The study followed by 3D modelling, prediction of linear B-cell epitope mapping, antigenicity, allergenicity, peptide properties, and immune simulation was carried out via a webserver. The 3D structure of the peptide was displayed through molecular visualization software. The results showed that the 23-mer peptides E1 'HPRYTGVKNIRDITLTEPGRGSD', F1 'NFIENRKGTQHN' 12-mer, M1 'YLKDQARLNS' 10-mer, N1 'PWVNDSIQPNWNNMTWQQWELQVRD' 25-mer, and O1 'KLQNSWNMGVQTO' can be used as a candidate polytope HIV-2 vaccine because it is recognized by B cells is an antigenic peptide with stable molecule, non-allergenic. The peptides trigger proliferation and activation of B cells to produces a humoral response and work as functionally protective antibody for neutralization of HIV-2.
 Key words: Acquired Immune Deficiency Syndrome, B-cell, Bioinformatics, Human Immunodeficiency Virus, Retrovirus

Identification of protective peptides of Fasciola hepatica-derived cathepsin L1 (FhCL1) in vaccinated sheep by a linear B-cell epitope mapping approach

BackgroundFasciolosis is one of the most important parasitic diseases of livestock. The need for better control strategies gave rise to the identification of various vaccine candidates. The recombinant form of a member of the cysteineprotease family, cathepsin L1 of Fasciola hepatica (FhCL1) has been a vaccine target for the past few decades since it has been shown to behave as an immunodominant antigen. However, when FhCL1 was used as vaccine, it has been observed to elicit significant protection in some trials, whereas no protection was provided in others.MethodsIn order to improve vaccine development strategy, we conducted a linear B-cell epitope mapping of FhCL1 in sheep vaccinated with FhCL1, FhHDM, FhLAP and FhPrx plus Montanide and with significant reduction of the fluke burden, sheep vaccinated with FhCL1, FhHDM, FhLAP and FhPrx plus aluminium hydroxide and with non-significant reduction of the fluke burden, and in unvaccinated-infected sheep.ResultsOur study showed that the pattern and dynamic of peptide recognition varied noticeably between both vaccinated groups, and that the regions 55–63 and 77–84, which are within the propeptide, and regions 102–114 and 265–273 of FhCL1 were specifically recognised only by vaccinated sheep with significant reduction of the fluke burden. In addition, these animals also showed significant production of specific IgG2, whereas a scarce non-significant production was observed in animals vaccinated with Aluminium hydroxide and no production was detected in infected control animals. ConclusionsWe have identified 42 residues of FhCL1 that contributed to protective immunity against infection with F. hepatica in sheep. Our results provide indications in relation to key aspects of the immune response. Given the variable outcomes of vaccination trials conducted in ruminants to date, this study adds new insights to improve strategies of vaccine development.

Designing a multi-epitope vaccine for cross-protection against Shigella spp: An immunoinformatics and structural vaccinology study

Shigellosis is a severe diarrheal disease with high mortality and morbidity rate. Until now, there is no approved vaccine against the disease. Therefore, the present study was planned to design a novel multi-epitope vaccine against Shigella spp., the causative agents of the disease based on the immunoinformatic tools. For this end, firstly seven conserved antigens of the bacteria, including IpaA, IpaB, IpaC, IpaD, OmpC, OmpF and VirG were selected. Then, linear B-cell epitope mapping of these proteins was carried out and top-ranked and shared epitopes were selected based on antigenicity, allergenicity, stability, toxicity and physicochemical properties for further analysis. In next step, B-cell derived T-cell epitopes were determined and appropriate epitopes were selected for incorporation into the final construct. Moreover, the selected epitopes and two mucosal adjuvants including ctxB and LT-IIc were joined using appropriate linkers. The three dimensional structure of the final construct was modeled and evaluated in term of structural quality and presence of conformational B-cell epitopes. Furthermore, binding affinity of the proposed vaccine to MHC I and II molecules were evaluated through molecular docking method using Hex 8.0. as well as the stability of the vaccine-MHC complexes was monitored by molecular dynamics method using the NAMD graphical user interface embedded in visual molecular dynamics. Finally, to evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. The results indicated that the proposed vaccine has high structural quality and binding affinity to both MHC I and II molecules. Moreover, molecular dynamics studies confirmed that the vaccine-MHC docked complexes were stable during simulation time. Animal study showed that the proposed protein is able to evoke mice's humoral immune response. In sum, the results suggested that the proposed candidate vaccine could be considered as a promising anti-shigellosis vaccine.

Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs.

In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, presenting a broad spectrum of clinical manifestations. Dogs are the main parasite reservoir in urban areas and canine cases precede human infection. Currently, A2 protein based Leish-Tec® vaccine is the only vaccine commercially available against CVL in Brazil. Considering that the main screening and confirmatory tests of canine infection are serological, it is possible that the antibody response elicited after vaccination interfere with diagnosis, leading to the inability to distinguish between vaccinated and infected animals. In order to identify the specific B-cell response induced after vaccination, A2 protein sequence was screened for main linear B-cell epitopes using in silico prediction (Bepipred) and immunological confirmation by ELISA. Three amino acid sequences were described as potential B-cell epitopes (SV11-SAEPHKAAVDV, PP16-PQSVGPLSVGPQSVGP, and VQ34-VGPLSVGPQSVGPLSVGPLSVGPQAVGPLSVGPQ). Specific IgG ELISAs were performed in sera of 12 immunized dogs living in non-endemic areas, followed for up to 1 year after immunization. The results were compared with those obtained in a group of 10 symptomatic and 10 asymptomatic CVL dogs. All predicted epitopes were confirmed as linear B-cell epitopes broadly recognized by sera from studied dogs. Total IgG ELISAs demonstrated distinct patterns of response between peptides in the immunized and CVL groups. VQ34 peptide was recognized by the majority of sera from vaccinated and symptomatic dogs, and increases after vaccination. PP16 induced low levels of specific IgG that increased 1 year after immunization. Interestingly, a low frequency of reactivity was found against SV11 in naturally infected dogs (symptomatic and asymptomatic), while 83.3% of vaccinated dogs presented positive responses 1 year after immunization. The two animals in the vaccinated group that did not respond to SV11 1 year after immunization presented positive serology both 30 days and 6 months after immunization. In summary, we identified three main linear B-cell epitopes in A2 based vaccine. Moreover, the humoral response against SV11 presented marked differences between infected and Leish-Tec vaccinated dogs, and should be further investigated, in large trials, to confirm its potential as a serological marker able to distinguish between infected and vaccinated dogs.

Detailed genetic analyses of the HN gene in human respirovirus 3 detected in children with acute respiratory illness in the Iwate Prefecture, Japan

We performed detailed genetic analyses of the partial hemagglutinin–neuraminidase (HN) gene in 34 human respirovirus 3 (HRV3) strains from children with acute respiratory illness during 2013–2015 in Iwate Prefecture, Japan. In addition, we performed analyses of the evolutionary timescale of the gene using the Bayesian Markov chain Monte Carlo (MCMC) method. Furthermore, we analyzed pairwise distances and performed selective pressure analyses followed by linear B-cell epitope mapping and N-glycosylation and phylodynamic analyses. A phylogenetic tree showed that the strains diversified at around 1939, and the rate of molecular evolution was 7.6 × 10−4 substitutions/site/year. Although the pairwise distances were relatively short (0.03 ± 0.018 [mean ± standard deviation, SD]), two positive selection sites (Cys544Trp and Leu555Ser) and no amino acid substitutions were found in the active/catalytic sites. Six epitopes were estimated in this study, and three mouse monoclonal antibody binding sites (amino acid positions 278, 281, and 461) overlapped with two epitopes belonging to subcluster C3 strains. Bayesian skyline plot analyses indicated that subcluster C3 strains have been increasing from 2004, whereas subcluster C1 strains have declined from 2004. Based on these results, Iwate strains were divided into two subclusters and each subcluster evolved independently. Moreover, our results suggested that some predicted linear epitopes (epitopes 3 and 5) are candidates for an HRV3 vaccine motif. To better understand the details of the molecular evolution of HRV, further studies are needed.

Automated High-Throughput Mapping of Linear B-Cell Epitopes Using a Statistical Analysis of High-Density Peptide Microarray Data.

Detailed information of antibodies' specificity is often missing or inadequate even for continuous (i.e., linear) epitopes. Recent developments in peptide microarray technology has enabled the synthesis of up to two million peptides per array thereby allowing linear peptide epitopes to be examined by a systematic amino acid substitution and positional scanning approach. This kind of analysis generates a very large body of data, which needs to be analyzed and interpreted in a robust and automated manner. Here, we describe a rational systematic approach to define linear antibody epitopes using ANOVA statistics to identify not only significant but also important residues involved in antibody recognition. This statistical approach can be used to perform a comprehensive linear epitope discovery. For polyclonal antibodies, this could be extended to entire proteins pinpointing critical residues for each epitope. We argue that the ANOVA analysis levels out issues of unknown peptide concentration/quality and unknown antibody titers leading to identification of epitopes that otherwise would be neglected if the evaluation was based merely on signal strength.

Linear B-cell epitope mapping of MAPK3 and MAPK4 from Leishmania braziliensis: implications for the serodiagnosis of human and canine leishmaniasis.

The correct and early identification of humans and dogs infected with Leishmania are key steps in the control of leishmaniasis. Additionally, a method with high sensitivity and specificity at low cost that allows the screening of a large number of samples would be extremely valuable. In this study, we analyzed the potential of mitogen-activated protein kinase 3 (MAPK3) and mitogen-activated protein kinase 4 (MAPK4) proteins from Leishmania braziliensis to serve as antigen candidates for the serodiagnosis of human visceral and tegumentary leishmaniasis, as well as canine visceral disease. Moreover, we mapped linear B-cell epitopes in these proteins and selected those epitopes with sequences that were divergent in the corresponding orthologs in Homo sapiens, in Canis familiaris, and in Trypanosoma cruzi. We compared the performance of these peptides with the recombinant protein using ELISA. Both MAPK3 and MAPK4 recombinant proteins showed better specificity in the immunodiagnosis of human and canine leishmaniasis than soluble parasite antigens and the EIE-leishmaniose-visceral-canina-bio-manguinhos (EIE-LVC) kit. Furthermore, the performance of this serodiagnosis assay was improved using synthetic peptides corresponding to B-cell epitopes derived from both proteins.

Identification of linear B epitopes of pertactin of Bordetella pertussis induced by immunization with whole and acellular vaccine

Pertussis is a serious infectious disease of the respiratory tract caused by the gram-negative bacteria Bordetella pertussis. There has been a reemergence of this disease within the population of several countries that have well established vaccination programs. Analyzes of clinical isolates suggest an antigenic divergence between the vaccine-based strains to the circulating strains. Although antibodies against P.69 are involved in the observed protective immunity, the sequences recognized as antigenic determinants in P.133, the precursor for P.69, P.3.4 and P.30, have not be determined. Here, the precise mapping of linear B-cell epitopes within the predicted P.133 pertactin sequences was accomplished using the SPOT-synthesis of peptide arrays onto cellulose membranes and screening with murine sera generated by vaccination with either the Pertussis cellular (miPc) or Pertussis acellular (miPa) vaccine. A total of 23 major epitopes were identified by sera from miPc vaccinated mice, while thirteen were identified by sera from miPa vaccinated mice. Of these epitopes, 12 epitopes were specifically identified by antibodies produced in response to the miPc vaccine and two were specific to the miPa vaccine. These epitopes were distributed throughout the pertactin sequence but a significant number were concentrated to the P.30 Prn segment. An analysis of the epitope correlation homologies indicated that the variations from the observed mutations in pertactin would not constitute a problem using these vaccines. In addition, the mapping of epitopes demonstrated a higher number of linear B-cell epitopes immunized with the Pc vaccine than the Pa vaccine.

Mapping B-cell epitopes for the peroxidoxin of Leishmania (Viannia) braziliensis and its potential for the clinical diagnosis of tegumentary and visceral leishmaniasis.

The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues 5AIDITRK11, 72RDELNVL78, 251KSKHNRREGY260, 269DENGIVLD276, and 341DETTLVRS348. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

Comprehensive mapping of West Nile virus (WNV)- and Japanese encephalitis virus serocomplex-specific linear B-cell epitopes from WNV non-structural protein 1

West Nile virus (WNV) non-structural protein 1 (NS1) elicits protective immune responses during infection of animals. WNV NS1-specific antibody responses can provide the basis for serological diagnostic reagents, so the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the conservation of these sites among the Japanese encephalitis virus (JEV) serocomplex members also needs to be defined. The present study describes the mapping of linear B-cell epitopes in WNV NS1. We screened eight NS1-specific mAbs and antisera (polyclonal antibodies; pAbs) from mice immunized with recombinant NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. The screen using mAbs identified four WNV-specific (including Kunjin virus) epitopes, located at aa 21-36, 101-116, 191-206 and 261-276 in WNV NS1. However, using pAbs, only three WNV-specific epitopes were identified, located at positions 101-116, 191-206 and 231-246. Two of these epitopes (aa 21-36 and 261-276) had different reactivity with mAbs and pAbs. The knowledge and reagents generated in this study have potential applications in differential diagnostics and epitope-based marker vaccine development for WNV and viruses of the JEV serocomplex.

Linear B-cell epitope mapping of neuraminidases of the 2009 A H1N1 viruses based on immunoinformatics

The 2009 A H1N1 viruses have spread throughout the world, as the viral neuraminidase (NA) is a receptor-destroying enzyme, removing sialic acid from carbohydrate chains attached to NA, and releasing the viruses from infected cells. In this study, the NA genes of Guangdong viruses were sequenced, then their B-cell epitopes were predicted, screened and assessed based on immunoinformatics. The antisera were raised in rabbits against five linear synthetic peptides spanning the NA protein of 2009 A H1N1. Five peptides, LR17, SS12, DP9, DS11 and DI14, respectively, are capable of eliciting neutralizing antibodies against 2009 A H1N1 in the in vitro microneutralization assay. DI14 was identified to be particularly potent in eliciting a neutralizing antibody titer comparable to that obtained with a whole virion-immunized serum. Immunization of rabbit with either five peptides triggered a 2009 A H1N1-specific antibody response as high as that obtained with the whole virion as immunogen. Alignment with databases showed that the amino acid residues of five epitope peptides are highly conserved among the NA sequences of 2009 A H1N1 strains isolated from the world. Altogether, these data indicate that LR17, SS12, DP9, DS11 and DI14 represent a promising candidate for an effective synthetic peptide-based vaccine against 2009 A H1N1 viruses.

Antigenic properties of the Leishmania infantum GRP94 and mapping of linear B-cell epitopes

In this study, we show that the glucose-regulated protein 94 (GRP94) from Leishmania infantum is a major target for humoral immune response during both visceral (VL) and mucocutaneous leishmaniasis (MCL). Also, the time-course of appearance of anti-GRP94 antibodies along the infection was analysed in hamsters ( Mesocricetus auratus) experimentally infected with L. infantum. Remarkably, the reactivity against the Leishmania GRP94 was observed very soon after challenge, at the time of appearance of a humoral response against Leishmania total proteins, and long before that the animals develop clinical symptoms of disease. Therefore, at least for golden hamsters, the reactivity against Leishmania GRP94 is an early marker of infection. Using a collection of synthetic peptides covering the complete sequence of the L. infantum GRP94, we have determined the main linear antigenic determinants recognised by sera from humans, dogs, and hamsters suffering from VL. Four synthetic peptides, located in highly divergent regions of the protein, were found to be immunodominants and recognised by VL sera of these three different origins. We discuss that the prominent antigenicity of Leishmania GRP94 may be related to recent findings involving GRP94, and other heat shock proteins, in relevant immune functions such as tumour immunogenicity and antigen presentation.

Mapping of linear B-cell epitopes on the major core protein p24 of human immunodeficiency virus type 1 (HIV-1).

Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes on the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were epitope mapped using a random fragment expression library representing the p17- and p24-encoding part of the gag open reading frame. F5-2 defined an epitope within amino acids (aa) 14-23 at the N-terminus of p24, and F5-4 defined an epitope within aa 153-174 in the C-terminus of p24. F5-16 did not recognize any of the fusion proteins produced by the expression library indicating that this MAb defines a true conformational epitope on p24. Since the N-terminus of p24 has been reported to be immunosilent in humans, 356 HIV-1 antibody-positive serum samples were tested for reactivity against the region of p24 defined by F5-2. More than one third of the samples recognized this region indicating that it is immunoreactive and, further, the presence of antibodies against this region was associated with a reduced CD4 cell count.

Mapping of linear B-cell epitopes of hepatitis B surface antigen

Eight monoclonal antibodies directed against the surface protein of hepatitis B virus (HBV) were tested using an epitope-mapping system (Pepscan) for characterizing antigenic domains. Four different amino acid sequences corresponding to linear epitopes were identified: one in pre-S1 corresponding to the sequence 29–36, two in pre-S2 corresponding to overlapping sequences 134–141 and 137–144, and one in the S region of the protein corresponding to the amino acid sequence 117–126.

Mapping of linear B-cell epitopes of the S2 subunit of pertussis toxin

The linear immunogenic and antigenic structure of the S2 subunit of pertussis toxin was investigated with synthetic peptides corresponding to regions of the protein sequence predicted to contain surface-exposed hydrophilic beta turns. Five peptides as peptide-bovine serum albumin conjugates were recognized by anti-pertussis toxin antiserum and were thus designated "immunogenic epitopes." Two prominent immunogenic epitopes were specified by peptides corresponding to sequences spanning R107-120 and R186-199, whereas peptides corresponding to residues R35-50 and R91-106 were only bound in low titer. Three peptides as thyroglobulin conjugates elicited antisera in rabbits that bound intact pertussis toxin by enzyme-linked immunosorbent assay and immunoblot. These peptides were designated "antigenic epitopes." The most prominent antigenic determinant was localized to the N-terminal end of the S2 sequence encompassing residue R1-7. Peptides R35-50 and R91-106 represented two minor antigenic epitopes. Antisera to two additional peptides corresponding to residues R134-149 and R186-199 recognized the S2 subunit only by Western blotting (immunoblotting). Only antiserum raised against peptide R91-106 also recognized the S3 subunit by Western blotting, indicating a marked antigenic and probably also structural difference between the two highly homologous subunits.