Identification of a new neutralization antigenic site on poliovirus coat protein VP2

Major neutralization antigenic sites have been previously mapped by us on VP1, the largest capsid protein of poliovirus type 1. Here we report the first identification of the primary sequence of a neutralization antigenic site on capsid protein VP2. Inspection of the amino acid sequence of VP2 led to the selection and synthesis of a peptide (n = 12) that, after linking to a carrier protein, induced an antiviral neutralizing antibody response in rabbits. The response was augmented by a single subsequent inoculation of intact virus; thus, the peptide was also capable of priming the production of neutralizing antibodies. These antibodies were directed only against the site specified by the synthetic peptide. Although the VP2-specific neutralization antigenic site appears not to be strongly immunogenic in the intact virion, it can nevertheless contribute to neutralization of poliovirus. This observation may be important for the development of peptide vaccines.     

Molecular basis for linkage of a continuous and discontinuous neutralization epitope on the structural polypeptide VP2 of poliovirus type 1.

We obtained neutralizing monoclonal antibodies against a continuous neutralization epitope on VP2 of poliovirus type 1 strain Mahoney by using a combined in vivo-in vitro immunization procedure. The antibody-binding site was mapped to amino acid residues within the peptide segment (residues 164 through 170) of VP2 by competition with synthetic peptide and sequencing of resistant mutants. Cross-neutralization of these mutants with another neutralizing monoclonal antibody revealed a linkage of the continuous epitope and a discontinuous neutralization epitope involving both loops of the double-loop structure of VP2 at the twofold axis on the surface of the virion.     

Identification and mapping of neutralizing epitopes of human parvovirus B19 by using human antibodies.

We identified and mapped the regions responsible for neutralization in the human parvovirus B19 structural protein by using region-specific human antibodies derived from seropositive blood donors. The region-specific antibodies were purified by using affinity columns coupled with synthetic peptides of the hydrophilic regions including the beta-turn structure deduced by the predicted secondary structure of VP2. Fifteen highly specific antibodies against the synthetic peptides were obtained. Ten of them were able to precipitate the radiolabeled virus. Six of them proved to be able to protect the colony-forming unit erythroid cells in human bone marrow cell cultures from injury by the virus. The sequences recognized by the six neutralizing antibodies were sites corresponding to amino acids 253 to 272, 309 to 330, 325 to 346, 359 to 382, 449 to 468, and 491 to 515 from the amino-terminal portion of VP2. These observations suggest that the neutralizing epitopes were distributed in the region from amino acid 253 in the amino-terminal portion of VP2 to the carboxyl terminus of VP2.     

Mapping of B-cell epitopes on the polypeptide chain of the Epstein-Barr virus major envelope glycoprotein and candidate vaccine molecule gp340.

The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.     

Priming for rotavirus neutralizing antibodies by a VP4 protein-derived synthetic peptide.

In the rotavirus SA11 surface protein VP4, the trypsin cleavage sites associated with the enhancement of infectivity are flanked by two amino acid regions that are highly conserved among different rotaviruses. We have tested the ability of synthetic peptides that mimic these two regions to induce and prime for a rotavirus neutralizing antibody response in mice. After the peptide immunization schedule, both peptides induced peptide antibodies, but neither was able to induce virus antibodies, as measured by an enzyme-linked immunosorbent assay or a neutralization assay. However, when the peptide-inoculated mice were subsequently injected with intact SA11 virus, a rapid and high neutralizing antibody response was observed in mice that had previously received the peptide comprising amino acids 220 to 233 of the VP4 protein. This neutralizing activity was serotype specific; however, this peptide was also able to efficiently prime the immune system of mice for a neutralizing antibody response to the heterotypic rotavirus ST3 when the ST3 virus was used for the secondary inoculation.     

Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice

The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M(1)ASLIYRQLL(10), a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1-10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection.     

Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii

Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28ΔN), C-terminal (rVP28ΔC) and middle (rVP28ΔM). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1–27) and middle region (residues 35–95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV.     

Candidate multi-epitope vaccines in aluminium adjuvant induce high levels of antibodies with predefined multi-epitope specificity against HIV-1

Some neutralizing epitopes on HIV-1 envelope proteins were identified to induce antibodies which could effectively inhibit the infection of different strains in vitro. But only very low levels of these antibodies were determined in the HIV-1 infected individuals. To increase the levels of protective antibodies in vivo, we suggested multi-epitope vaccine as a new strategy to induce high level of neutralization antibodies with predefined multi-epitope specificity. A synthesized epitope peptide MP (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) containing three neutralizing epitopes (GPGRAFY, ELDKWA, RILAVERYLKD) was conjugated to carrier protein KLH, and then used for immunization in mouse together with aluminium adjuvant or Freund's adjuvant (FA). The candidate MP-KLH multi-epitope vaccine in aluminium adjuvant could induce antibody response very strongly to the epitope peptide C-(RILAVERYLKD-G)2 and the immunosuppressive peptide (P1) (LQARILAVERYLKDQQL) (antibody titer: 1:51200), strongly to the epitope peptide C-(ELDKWA-G)4 and the C-domain peptide (P2) (1:12800), and moderately to the epitope peptide C-(GPGRAFY)4 and the V3 loop peptide (1:1600). The immunoblotting analysis demonstrated that the antibodies in sera could recognize P1, P2, V3 loop peptides and rsgp41 (aa 539-684). These results are similar with that in the case of PI-BSA in FA, and suggest that the multi-epitope vaccine in aluminium could induce high levels of antibodies of predefined multi-epitope specificity, which provides experimental evidence for the new strategy to develop an effective neutralizing antibody-based multi-epitope vaccine against HIV-1.     

Synthetic Peptide-based Vaccine and Diagnostic System for Effective Control of FMD

We have designed synthetic peptides corresponding to two different regions of the genome of foot-and-mouth disease virus (FMDV) that are effective as (a) a vaccine or (b) a diagnostic reagent which differentiates convalescent from vaccinated animals, respectively. The peptide vaccine is based on a sequence from the prominent G-H loop of VP1, one of the four capsid proteins. The sequence was optimized by the inclusion of a cyclic constraint and adjoining sequences, and broader immunogenicity was obtained by the incorporation of consensus residues at hypervariable positions. The peptide also included a promiscuous T-helper epitope for effective immunogenicity in outbred populations of large animals.The diagnostic reagent, a peptide based on non-structural (NS) protein 3B, is used in immuno-assays for the detection of antibodies. Antibodies to this NS protein are present in the sera of infected animals but not in the sera of vaccinated animals. The VP1 peptide can be used in complementary immuno-assays for confirmation of NS test results and to monitor for vaccination. This system for differential diagnosis is important to establish the disease-free status of a country.     

Identification of a functional domain of human granulocyte colony-stimulating factor using neutralizing monoclonal antibodies.

Human granulocyte colony-stimulating factor (G-CSF) is a hemopoietic growth factor that is being used successfully to treat various forms of neutropenia. To define functionally important regions of G-CSF, we have prepared 37 monoclonal anti-G-CSF antibodies and mapped the regions of G-CSF recognized by different antibody groups. Antibodies recognizing similar epitopes were identified by competition assays, neutralization assays, conformation dependence and cross-reactivity with canine G-CSF. Seven of eight neutralizing antibodies fell into two related epitope groups and were conformation-dependent. The eighth was unrelated and conformation-independent. Peptides of G-CSF were generated by chemical or enzymatic digestion and tested for antibody reactivity. One of the neutralizing antibodies (LMM351) recognized a small, disulfide-bonded peptide from the V8 protease digest (residues 34-46). A synthetic peptide (residues 20-58) was recognized by all the neutralizing antibodies, implicating this disulfide-bonded loop in receptor binding. The epitopes recognized by nonneutralizing antibodies were found throughout G-CSF. Thus, regions of G-CSF that are not involved in receptor binding have also been defined. A CNBr peptide (residues 1-121) had greatly reduced biological activity, indicating that the COOH terminus is required for receptor binding. We predict that residues 20-46 and the COOH terminus bind to the G-CSF receptor.     

Antibodies to the Buried N Terminus of Rhinovirus VP4 Exhibit Cross-Serotypic Neutralization

Development of a vaccine for the common cold has been thwarted by the fact that there are more than 100 serotypes of human rhinovirus (HRV). We previously demonstrated that the HRV14 capsid is dynamic and transiently displays the buried N termini of viral protein 1 (VP1) and VP4. Here, further evidence for this “breathing” phenomenon is presented, using antibodies to several peptides representing the N terminus of VP4. The antibodies form stable complexes with intact HRV14 virions and neutralize infectivity. Since this region of VP4 is highly conserved among all of the rhinoviruses, antiviral activity by these anti-VP4 antibodies is cross-serotypic. The antibodies inhibit HRV16 infectivity in a temperature- and time-dependent manner consistent with the breathing behavior. Monoclonal and polyclonal antibodies raised against the 30-residue peptide do not react with peptides shorter than 24 residues, suggesting that these peptides are adopting three-dimensional conformations that are highly dependent upon the length of the peptide. Furthermore, there is evidence that the N termini of VP4 are interacting with each other upon extrusion from the capsid. A Ser5Cys mutation in VP4 yields an infectious virus that forms cysteine cross-links in VP4 when the virus is incubated at room temperature but not at 4°C. The fact that all of the VP4s are involved in this cross-linking process strongly suggests that VP4 forms specific oligomers upon extrusion. Together these results suggest that it may be possible to develop a pan-serotypic peptide vaccine to HRV, but its design will likely require details about the oligomeric structure of the exposed termini.Rhinoviruses are the major causative agents of the common cold and cost the United States economy approximately $40 billion per year (6). Therefore, it is of great interest to prevent or ameliorate the symptoms of the common cold. The rhinovirus genus is a member of the picornavirus family and is characterized by nonenveloped capsid with a diameter of ∼300 Å containing a single-stranded, plus-sense RNA genome (19). Other members of the picornavirus family include foot-and-mouth disease virus, poliovirus, encephalomyocarditis virus, and hepatitis A virus. The capsids exhibit pseudo T = 3 icosahedral symmetry and are composed of 60 copies of the four capsid proteins VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 have an eight-stranded antiparallel beta-barrel motif structure and form the outer surface of the capsid, while VP4 lies at the interface between the capsid and the interior genomic RNA (22). VP4 is approximately 70 amino acids in length and is myristoylated at the N terminus (3, 14).Antibodies are the major line of defense against picornavirus infections. In the case of human rhinovirus 14 (HRV14), a number of studies have been performed to detail the antibody recognition and neutralization processes (25). While it had been long suggested that antibodies neutralize viral infectivity by inducing large conformational changes in the capsid, both cryo-transmission electron microscopy (cryo-TEM) (2, 28) and crystallographic analysis (27) clearly demonstrated that this was not the case. Further, it was shown that antibody recognition is more plastic than previously thought in that it is able to bind into the relatively narrow receptor-binding region of the canyon (27). These results suggested that the major in vivo role of antibodies is to bind to virion and work synergistically with other immune system components (26). This hypothesis has gained further support from studies of other pathogens (1) and implies that vaccines need only to elicit antibodies that bind to the authentic pathogen with high affinity.While these results simplified the goal of creating a synthetic vaccine by focusing on capsid recognition rather than possible antibody-induced conformational changes, developing synthetic vaccines against all 100 serotypes of HRV remains a daunting task. As shown in the structures of HRV14/antibody complexes, the antibodies make extensive contacts with the surface of the capsid that is not limited to a single antigenic loop (2, 27). Further evidence for this extensive contact is that antibodies to peptides corresponding to antigenic NIm loops fail to neutralize the virions (17, 29), and antibodies raised against intact capsids do not bind effectively to peptides corresponding to NIm-IA loop (T. J. Smith, unpublished results). One notable exception is the case of HRV2, where there is cross-reactivity between the NIm-II site of the virion and a synthetic peptide (30). Nevertheless, developing a repertoire of peptides representing the entire antigenic ensemble of HRVs is not only impractical but also unlikely to elicit neutralizing antibodies.All of the studies described above were performed with the antibodies that were raised against intact particles or to peptides representing epitopes that reside on the outer surface of the capsid. In the case of poliovirus, however, antibodies were raised against VP4 and the N termini of VP1 of poliovirus serotype I (15, 21). It was shown that these antibodies are capable of neutralizing the virion despite the fact that those portions of the capsid protein are buried in the interior of the capsid at the capsid-RNA interface (8). These results suggested that the poliovirus capsid was more dynamic than indicated by the crystal structure and that these termini are presented to the exterior of the virion in a temperature-dependent and reversible manner. While the role of capsid dynamics in the viral life cycle was not clear, it was suggested that the N termini of VP1 and VP4 might facilitate cell membrane attachment and subsequent entry of the virus into the host cell (3, 4).More recently, evidence for capsid dynamics has been found in other viruses as well. In the cases of swine vesicular disease virus (10) and coxsackievirus A9 (18), antibodies were raised against the whole virus in pigs and rabbits, respectively. These polyclonal antibodies demonstrated a strong reaction to the peptides corresponding to the N termini of VP1 and VP3 of swine vesicular disease virus and coxsackievirus A9, respectively. In a similar study, antibodies from the plasma of patients suffering from type I diabetes were found to target VP4 protein of coxsackievirus B3, again suggesting the exposure of VP4 peptide during coxsackievirus infection (23). These results imply that capsid “breathing” may be a phenomenon common to many proteinaceous capsids.Using a very different approach, the dynamic nature of HRV14 was analyzed using limited proteolysis and mass spectrometry (matrix-assisted laser desorption ionization [MALDI]) analyses (14). In these experiments, the virus was treated with both matrix-bound and soluble forms of trypsin for various periods of time, and the resulting proteolytic fragments were identified by MALDI. Surprisingly, the N termini of VP4 and VP1 were found to be the most proteolytically sensitive portions of the capsid in spite of being buried inside the viral capsid. As an additional control, the antiviral “WIN” compounds, which had been previously shown to stabilize the virions against thermal and acid denaturation, were added during digestion. While these WIN compounds did not affect the intrinsic proteolytic activity of trypsin, they nearly completely protected the VP1 and VP4 termini from proteolysis for an extended period. Together, these results suggested that HRV14 is transiently exposing these termini in a “breathing” process and that the empty hydrophobic drug-binding region apparently plays an important role in facilitating these dynamics.In this study we further examined HRV14 capsid dynamics by raising polyclonal antibodies against several peptides representing the N termini of VP1 and VP4. In these experiments, only the antibodies against the VP4 N terminus were found to successfully neutralize viral infectivity in vitro. Further, we demonstrate that the HRV14 VP4 antiserum cross-reacts with other serotypes of rhinovirus (HRV16, and HRV29), which is likely due to the high degree of conservation of VP4. Antibody neutralization closely parallels the MALDI analysis in that antibody neutralization and proteolysis are enhanced at 37°C in the case of HRV16 whereas the elevated temperatures are not required for either phenomenon in the cases of HRV14 and HRV29. Epitope mapping of the N-terminal 30 residues of VP4 suggests that it adopts a nonlinear conformation, and this is further substantiated by results showing that all of the copies of VP4 in the Ser5Cys HRV14 mutant at room temperature form cysteine cross-linked dimers. This cysteine cross-link does not form at 4°C, suggesting that capsid breathing is essential for VP4 exposure and interactions. Since VP4 dimerization does not affect viral infectivity, it seems likely that VP4 extrusion is a normal part of the cell attachment and entry process of rhinovirus. Together, these results suggest that VP4 might be useful as a pan-serotypic rhinovirus vaccine, but it seems likely that better understanding of the VP4 oligomeric structure will be necessary for further optimization.     

A structure-based approach to a synthetic vaccine for HIV-1

The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH --> O=CRNH) with a hydrazone link (N-N=CH-CH(2)CH(2)) and scan constrained peptides for fit with neutralizing monoclonal antibodies (MAbs). To explore this approach, a V3 MAb 58.2 that potently neutralizes T-cell lab-adapted HIV-1(MN) was used to identify a cyclic peptide, [JHIGPGR(Aib)F(D-Ala)GZ]G-NH(2) (loop 5), that binds with >1000-fold higher affinity than the unconstrained peptide. NMR structural studies suggested that loop 5 stabilized beta-turns at GPGR and R(Aib)F(D-Ala) in aqueous solvent implying considerable conformational mimicry of a Fab 58.2 bound V3 peptide determined by X-ray crystallography [Stanfield, R. L. et al. (1999) Structure 142, 131-142]. Rabbit polyclonal antibodies (PAbs) generated to loop 5 but not to the corresponding uncyclized peptide bound the HIV-1(MN) envelope glycoprotein, gp120. When individual rabbit antisera were scanned with linear and cyclic peptides, further animal-to-animal differences in antibody populations were characterized. Loop 5 PAbs that most closely mimicked MAb 58.2 neutralized HIV-1(MN) with similar potency. These results demonstrate the remarkable effect that conformation can have on peptide affinity and immunogenicity and identify an approach that can be used to achieve these results. The implications for synthetic vaccine and HIV-1 vaccine research are discussed.     

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.     

Analysis of neutralizing epitopes on foot-and-mouth disease virus.

For the investigation of the antigenic determinant structure of foot-and-mouth disease virus (FMDV), neutralizing monoclonal antibodies (MAbs) against complete virus were characterized by Western blot (immunoblot), enzyme immunoassay, and competition experiments with a synthetic peptide, isolated coat protein VP1, and viral particles as antigens. Two of the four MAbs reacted with each of these antigens, while the other two MAbs recognized only complete viral particles and reacted only very poorly with the peptide. The four MAbs showed different neutralization patterns with a panel of 11 different FMDV strains. cDNA-derived VP1 protein sequences of the different strains were compared to find correlations between the primary structure of the protein and the ability of virus to be neutralized. Based on this analysis, it appears that the first two MAbs recognized overlapping sequential epitopes in the known antigenic site represented by the peptide, whereas the two other MAbs recognized conformational epitopes. These conclusions were supported and extended by structural analyses of FMDV mutants resistant to neutralization by an MAb specific for a conformational epitope. These results demonstrate that no amino acid exchanges had occurred in the primary antigenic site of VP1 but instead in the other coat proteins VP2 and VP3, which by themselves do not induce neutralizing antibodies.     

Generation of neutralizing monoclonal antibodies against Enterovirus 71 using synthetic peptides

Enterovirus 71 (EV71) has led to recent outbreaks of hand, foot and mouth disease (HFMD) in China, resulting in high mortality. In this study, several monoclonal antibodies were generated by immunizing mice with two synthetic peptides, SP55 and SP70, containing amino acids 163-177 and 208-222 of VP1. The specificities of the anti-EV71 peptide monoclonal antibodies were confirmed by Western blot analysis and immunocytochemistry against EV71 virus. Most importantly, we have identified a monoclonal antibody, clone 22A12, which shows strong neutralizing activity against EV71 in an in vitro neutralization assay. Because there is no vaccine available and treatment is very limited, mouse anti-EV71 monoclonal antibody, clone 22A12, could be a promising candidate to be humanized and used for treatment of EV71 infection.     

Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus.

The amino acid sequences recognized by monoclonal antibodies (MAbs) specific for the antigenic site IV of the spike protein S of transmissible gastroenteritis virus were analyzed by PEPSCAN. All MAbs of group IV recognized peptides from the S region consisting of residues 378 to 390. In addition, the neutralizing MAbs (subgroup IV-A) also bound to peptides from the region consisting of residues 1173 to 1184 and to several other peptides with a related amino acid composition. The contribution of the individual residues of both sequences to the binding of a MAb was determined by varying the length of the peptide and by a consecutive deletion or replacement of parental residues by the 19 other amino acids. The sequence consisting of residues 326 to 558, tested as part of a cro-beta-galactosidase hybrid protein, was antigenic, but the sequence consisting of residues 1150 to 1239 was not. Furthermore, antibodies raised in rabbits against the peptide SDSSFFSYGEIPFGN (residues 377 to 391), but not those raised against the peptide VRASRQLAKDKVNEC (residues 1171 to 1185), recognized the virus and had neutralizing activity. We infer that the epitope of the neutralizing MAbs is composite and consists of the linear sequence SFFSYGEI (residues 380 to 387) with contributions of A, D, K, N, Q, or V residues from other parts of the S molecule. The complex epitope was simulated by synthesizing peptides in which the sequences consisting of residues 380 to 387 and 1176 to 1184 were combined. MAbs of subgroup IV-A recognized the combination peptides two to six times better than the individual sequences. These results may offer prospects for the development of an experimental vaccine.     

A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1.

Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.     

Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.     

Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody

The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the K(d) values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH(2) could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope.     

Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.