Effect of chain length of HAV-VP3 synthetic peptides on its interaction with biomembrane models.

Shorter analogues of a continuous epitope of hepatitis A virus, VP3(110-121) peptide, failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. To better understand the influence of the structural properties of this 12-mer peptide epitope on its biological activity, the interaction of smaller peptide analogues with phospholipid biomembrane models was investigated by a combination of spectroscopic and biophysical techniques. In this article we describe our findings concerning the surface activity and the interaction of peptides with simple mono- and bilayer membranes composed of a zwitterionic phospholipid (dipalmitoyl phosphatidylcholine, DPPC), an anionic phospholipid (dipalmitoyl phosphatidylglicerol, DPPG), or a DPPC/DPPG mixture. The results indicate that the net negative charge of the peptide is in some way responsible of the specific interactions between VP3(110-121) and membrane phospholipids, and necessary to induce beta-type conformations upon vesicle interaction.

Generation of monoclonal antibodies specifically directed against the proximal zinc finger of HIV type 1 NCp7.

The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.

Use of linear and multiple antigenic peptides in the immunodiagnosis of acute hepatitis A virus infection.

The reactivities of two panels of anti-HAV human sera from geographically distinct areas (Chile and Spain) to synthetic peptides from the VP1, VP2 and VP3 hepatitis A virus capsid proteins were examined by an enzyme-linked immunosorbent assay (ELISA) procedure. Two and four branched multiple antigenic peptides (MAPs) and palmitoylated peptides were compared with free synthetic sequences for the detection of IgM anti-HAV antibodies in the two panels of human sera. Our results showed that acute hepatitis A patient sera recognized preferentially homogeneous two branched MAPs and palmitic acid conjugated peptides. The palmitoyl-derived VP3(110-121) peptide and the corresponding dimeric MAP were the most sensitive and appropriate for serological studies of HAV-infected patients by ELISA, sensitivity and specificity being higher than 90% and 95%, respectively. These peptide-based tests open up new avenues in the development of peptide-based immunosorbent assays for the detection of acute HAV disease.

Phospholipid-model membrane interactions with branched polypeptide conjugates of a hepatitis A virus peptide epitope.

To establish correlation between structural properties (charge, composition, and conformation) and membrane penetration capability, the interaction of epitope peptide-carrier constructs with phospholipid model membranes was studied. For this we have conjugated a linear epitope peptide, (110)FWRGDLVFDFQV(121) (110-121), from VP3 capside protein of the Hepatitis A virus with polylysine-based branched polypeptides with different chemical characteristics. The epitope peptide elongated by one Cys residue at the N-terminal [C(110-121)] was attached to poly[Lys-(DL-Ala(m)()-X(i)())] (i < 1, m approximately 3), where x = ø(AK), Ser (SAK), or Glu (EAK) by the amide-thiol heterobifunctional reagent, 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester. The interaction of these polymer-[C(110-121)] conjugates with phospholipid monolayers and bilayers was studied using DPPC and DPPC/PG (95/5 mol/mol) mixture. Changes in the fluidity of liposomes induced by these conjugates were detected by using two fluorescent probes 1,6-diphenyl-1,3, 5-hexatriene (DPH) and sodium anilino naphthalene sulfonate (ANS). The binding of conjugates to the model membranes was compared and the contribution of the polymer component to these interactions were evaluated. We found that conjugates with polyanionic/EAK-[C(110-121)] or polycationic/SAK-[C(110-121)], AK-[C(110-121)]/character were capable to form monomolecular layers at the air/water interface with structure dependent stability in the following order: EAK-[C(110-121)] > SAK-[C(110-121)] > AK-[C(110-121)]. Data obtained from penetration studies into phospholipid monolayers indicated that conjugate insertion is more pronounced for EAK-[C(110-121)] than for AK-[C(110-121)] or SAK-[C(110-121)]. Changes in the fluorescence intensity and in polarization of fluorescent probes either at the polar surface (ANS) or within the hydrophobic core (DPH) of the DPPC/PG liposomes suggested that all three conjugates interact with the outer surface of the bilayer. Marked penetration was documented by a significant increase of the transition temperature only with the polyanionic compound/EAK-[C(110-121)]. Taken together, we found that the binding/penetration of conjugates to phospholipid model membranes is dependent on the charge properties of the constructs. Considering that the orientation and number of VP3 epitope peptides attached to branched polypeptides were almost identical, we can conclude that the structural characteristics (amino acid composition, charge, and surface activity) of the carrier have a pronounced effect on the conjugate-phospholipid membrane interaction. These observations suggest that the selection of polymer carrier for epitope attachment might significantly influence the membrane activity of the conjugate and provide guidelines for adequate presentation of immunogenic peptides to the cells.

Gag protein epitopes recognized by CD4(+) T-helper lymphocytes from equine infectious anemia virus-infected carrier horses.

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.

Promiscuous T-cell recognition of a rubella capsid protein epitope restricted by DRB1*0403 and DRB1*0901 molecules sharing an HLA DR supertype.

Two T cell clones derived from different donors with HLA-DRB1*0403 or DRB1*0901 phenotype recognize a rubella capsid peptide, C(265-273) in the context of several different HLA-DR molecules in addition to DRB1*0403 and DRB1*0901. All DR molecules restricting the T-cell clones have in common residues, R or Q at position beta 70, R at position beta 71, and E at position beta 74 in pocket '4' of the DR peptide binding groove, suggesting that a DR subregion structure or supertype, "Q/RRE" underlies the promiscuous T-cell recognition of this peptide. Single amino acid substituted analogs of peptide C(263-275) at anchor position 4 for natural residue R were tested for their ability to induce clonal T-cell cytotoxic responses. The results indicated that a positively charged residue, R or K, was required for T-cell recognition, suggesting a possible mechanism of electrostatic interactions between the negatively charged residue E at position beta 74 of these DR molecules and the positively charged residue at anchor position 4 of the peptide in T-cell recognition.

Viral coat protein peptides with limited sequence homology bind similar domains of alfalfa mosaic virus and tobacco streak virus RNAs.

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.

Physico-chemical characterization of liposomes with covalently attached hepatitis A VP3(101-121) synthetic peptide.

The covalent conjugation of a 20-mer peptide belonging to the VP3 capsid protein of hepatitis A virus to the surface of preformed liposomes was investigated. Three different bonds (disulfide, thioether and amide) were established between the peptide sequence and liposomes bearing at their surface appropriate reactive groups. The effect of the relative concentration of the N-[4-(p-maleimidophenyl)butyryl]dipalmitoylphosphatidylethanolamine anchor in liposomes on stability during coupling of the peptide sequence was studied. The interaction of the three liposomal preparations with phospholipids in a biomembrane model system, monolayers at the air-water interface, is also reported. The results showed that although the peptides associate with liposomes in similar yields for the three strategies studied, differences can be observed when their interaction with phospholipid monolayers composed of dipalmitoylphosphatidylcholine is analysed.

Synthesis and biological activities of fluorescent acridine-containing HIV-1 nucleocapsid proteins for investigation of nucleic acid-NCp7 interactions.

Specific interactions between the 72-amino acid nucleocapsid protein NCp7 of the human immunodeficiency virus, type 1 and the genomic RNA are essential for virus replication. Studies on the mechanism of action of NCp7 require a direct visualization of its complexes with nucleic acids and the determination of binding affinities. To facilitate these investigations, fluorescent NCp7 derivatives were developed by introduction in the NCp7 sequence of a non-natural amino acid, (S)-beta-(9-acridinyl)alanine (Aca) obtained by a chiral synthetic method. Three fluorescent NCp7 derivatives were obtained by introducing this amino acid at different positions. As shown by NMR, the three-dimensional structure of NCp7 is not altered by introduction of Aca. The fluorescent peptides were found to be as potent as their precursors in interacting with nucleic acids and in promoting HIV-1 genomic RNA dimerization. Moreover, because of their fluorescent properties, these NCp7s can be used at submicromolar concentrations to directly visualize and quantify protein-nucleic acid interactions in solution or after gel electrophoresis. This could facilitate the development of new antiviral agents aimed at inhibiting the functions of NCp7 and studies on the intracellular traffic of NCp7 within the preintegration complex.

In vivo T helper cell response to retro-inverso peptidomimetics.

Peptide analogues containing reversed peptide bonds between each residue along the peptide sequence (retro-inverso modification) have been analyzed for their antigenic and in vivo immunogenic properties in the MHC II and Th cell response context. Two antigenic peptides were selected for this study, namely peptide 103-115 of poliovirus VP1, which is involved in the production of Abs that neutralize the infectivity of the virus, and peptide 435-446 from the third constant region of mouse heavy chain IgG2a allopeptide gamma 2ab, which mimics a corneal Ag implicated in autoimmune keratitis. In a competition assay performed in vitro using reference hybridomas of known MHC class II restriction, both retro-inverso analogues bound (although more weakly in our test) to I-Ad and/or I-Ed class II molecules. However, in both cases, this lower affinity was apparently largely compensated in vivo, as a T cell response (with IL-2 secretion), equivalent to that obtained with the wild-type peptides, was observed following immunization of BALB/c mice with the retro-inverso analogues. Moreover, these T cells proliferated and produced IL-2 in response to the cognate peptides. It is concluded that the T cell receptors of T cells primed in vivo with the retro-inverso analogues readily cross-react with parent and retro-inverso analogue-MHC complexes. The approach of using pseudopeptides containing changes involving the backbone, and not the orientation of side chains, may thus be promising to design potent immunogens for class II-restricted T cells.

A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants.

A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.

Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler's virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus-induced immunologically mediated demyelinating disease (TMEV-IDD) in susceptible mice provides a relevant infectious model for multiple sclerosis. Previously, we have identified six major linear antibody epitopes on the viral capsid proteins. In this study, we utilized fusion proteins containing individual capsid proteins and synthetic peptides containing the linear antibody epitopes to determine the potential role of antibody response in the course of virus-induced demyelination. Preimmunization of susceptible mice with VPI and VP2 fusion proteins, but not VP3, resulted in the protection from subsequent development of TMEV-IDD. Mice free of clinical symptoms following preimmunizations with fusion proteins displayed high levels of antibodies to the capsid proteins corresponding to the immunogens. In contrast, the level of antibodies to a particular linear epitope, A1C (VP1(262-276)), capable of efficiently neutralizing virus in vitro increased with the progression of disease. Further immunization with synthetic peptides containing individual antibody epitopes indicated that antibodies to the epitopes are differentially effective in protecting from virus-induced demyelination. Taken together, these results suggest that antibodies to only certain linear epitopes are protective and such protection may be restricted during the early stages of viral infection.

Inhibition of cell adhesion to the virus by synthetic peptides of fiber knob of human adenovirus serotypes 2 and 3 and virus neutralisation by anti-peptide antibodies.

The fiber knob of adenovirus (Ad) causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s) several synthetic peptides derived from Ad2 and Ad3 fiber head sequences and their antisera were tested for interference with virus attachment to HeLa and FL cells and cell adhesion to viruses. The anti-peptide sera were also evaluated in ELISA and virus neutralisation test. Ad2 (of subgroup C) and Ad3 (of subgroup B) attachment was not significantly inhibited by peptides corresponding to the amino acid residues 535-554, 555-573, 562-582 of Ad2 fiber or 210-225, 267-283, 291-306 and 300-319 of Ad3 fiber. However, microplate pre-adsorbed Ad3 fiber residues 210-225 and 267-283 could bind FL and HeLa cells, and 1 mg/ml of Ad3 fiber residues 267-283 inhibited the cell adhesion to Ad3 virus to approximately 90%. This peptide may participate in the receptor binding site of Ad3 fiber. ELISA reactive anti-peptide antibodies against the homologous peptide and virus did not significantly reduce the cell adhesion to the immobilised virus or the virus attachment to cells, but in the neutralisation assay antibodies raised to Ad2 fiber residues 555-573 and 562-582 and Ad3 fiber residues 210-225 caused neutralisation of the homologous virus at serum dilutions of 1:500 and 1:32, respectively. The corresponding peptides and one further peptide of Ad2 fiber and two of Ad3 fiber seem to contain neutralisation epitopes.

A study of the cellular immune response to enteroviruses in humans: identification of cross-reactive T cell epitopes on the structural proteins of enteroviruses.

We have attempted to extend our understanding of the enteroviral cross-reactive T cell response in humans. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated in vitro with six different serotypes of enterovirus and 15 synthetic peptides representing conserved regions in the four structural proteins of these viruses. Upon challenge with different antigens, PBMC from donors responded specifically with proliferation and production of interferon-gamma (IFN-gamma). In contrast, synthesis of interleukin-4 (IL-4) or IL-10 was not detected. A T cell response to each enterovirus serotype was recorded in all individuals even though not all individuals had serum neutralizing antibody against each virus. These data confirmed previous findings that human T cells recognize enteroviral cross-reactive epitopes. Analysis of the peptide-induced IFN-gamma production and proliferative response showed that the cross-reactive T cell epitopes are localized mainly in capsid protein VP2 and VP3 and to a lesser extent in VP1. Surprisingly, T cell epitopes were not identified in the most conserved structural protein of enterovirus, VP4. Immune responses were mediated by CD4+ T cells in association with MHC class II molecules. The sources of IFN-gamma in response to the most immunodominant cross-reactive T cell epitopes were CD4+, CD8+ and NK cells. The two latter subsets produced IFN-gamma provided CD4+ T cells were present. Since T helper 1 (Th1) cells can mediate an in vivo protective immune response against poliovirus infection in mice, our novel findings in humans merit further detailed characterization of T cells that recognize the enteroviral cross-reactive T cell epitopes.

Chemically synthesised human immunodeficiency virus P7 nucleocapsid protein can self-assemble into particles and binds to a specific site on the tRNA(Lys,3) primer.

The zinc-bound form of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, p7, aggregates into particles visible by electron microscopy. The HIV primer tRNA(Lys,3) forms similar high molecular weight complexes with p7 that are also detected by gel mobility shift assays. RNA oligonucleotides of the three stem-loop structures in tRNA(Lys,3) were assayed for the competitive inhibition of p7-tRNA(Lys,3) binding by the intensities of free tRNA(Lys,3) bands on native gels. This reveals that the p7 binds specifically to the central domain of tRNA(Lys,3) where the D and T psi C loops come together, but not the anticodon stem-loop.

Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy.

The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be critical in various NCp7 functions, especially in the encapsidation process. To further understand the central role of zinc binding to NCp7, we closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivatives. To this end, the fluorescence of wither the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, respectively. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with 2.8 x 10(14) M-1 binding constant about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this negative cooperativity was shown to be related to the spatial proximity of the zinc-saturated finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were critical and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding constants to both sites. Taken together, our data provide further evidence for the nonequivalence of the two NCp7 finger motifs.

Use of a constrain phage displayed-peptide library for the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7).

It has been shown that peptide libraries are powerful tools for the identification of peptides showing new binding specificity. This technology was applied to the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7). Three different prolin reach peptide sequences, interacting with NCp7, were isolated, from a constrained phage displayed-peptide library of 10(8) independent clones. The three peptide sequences, isolated from the peptide library, were shown to bind NCp7 in the region 30-52. Moreover, two of them share the PP-(D/E)R consensus sequence.

Solid phase synthesis and immunogenicity of a VP3 peptide from hepatitis A virus.

The synthesis of a peptide belonging to the VP3 capsid protein of Hepatitis A virus has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of methoxytrimethylbenzenesulphonyl (Mtr) and pentamethylchromansulphonyl (Pmc) as arginine side-chain protecting groups in the presence of tryptophan without lateral protection or protected with t-Boc is discussed. The synthetic VP3 peptide has been administered to mice in different forms: (i) free, (ii) coupled to keyhole limpet hemocyanin, (iii) encapsulated in multilamellar (MLV) liposomes, and (iv) incorporated to a tetrameric branched lysine core. The immune response induced by these preparation is reported.

Antibodies to EYRKK vesicular stomatitis virus-related peptide account only for a minority of anti-Ro60kD antibodies.

Previous studies demonstrated a possible antigenic relation between the carboxyl terminal portion of anti-Ro60kD autoantigen and a nucleocapsid protein (N) of vesicular stomatitis virus (VSV). In order to investigate whether anti-Ro60kD autoantibodies react with the VSV homologous region of the Ro60kD protein we synthesized, according to Merrifield's method, the EYRKKMDI octapeptide (8p) sharing a common sequence with the N protein of VSV. Sera from 61 patients with autoimmune rheumatic diseases (34 systemic lupus erythematosus (SLE), 21 Sjörgren's syndrome (SS) and six rheumatoid arthritis (RA)) as well as 59 from normal blood donors were tested for the presence of anti-Ro60kD autoantibodies by ELISA and immunoblot (IB) and anti-8p antibodies by ELISA. Antibodies to 8p were found in 9/31 of anti-Ro60kD IB-positive sera, 5/30 of anti-Ro60kD-negative sera and 2/59 of normal control sera. The concordance between the anti-8p ELISA and the anti-Ro60kD IB was very poor (chi 2 = 0.71, P = 0.4) in contrast to the anti-Ro60kD ELISA and the anti-Ro IB (chi 2 = 27.6, P = 10(-7)). Subsequent affinity purification of the anti-8p antibodies from a strong positive anti-8p and anti-Ro60kD SLE serum yielded 95% depletion of the anti-8p activity and 37% reduction of the anti-Ro60kD activity. Inhibition assays with the affinity-purified anti-8p antibodies demonstrated that the octapeptide gave 94.5% inhibition of the anti-Ro60kD activity, while Ro60kD protein led to 42.3% inhibition of the anti-8p. Preincubation of the serum with the octapeptide produced 4% inhibition of anti-Ro60kD ELISA. These results indicate that the anti-8p antibodies account only for a minority of the anti-Ro60kD autoantibodies.

Effective induction of neutralizing antibodies with the amino terminus of VP2 of canine parvovirus as a synthetic peptide.

Fourteen synthetic peptides corresponding to previously mapped antigenic sites in VP2 of canine parvovirus (CPV) were used for immunization of rabbits to identify antiviral properties favourable for inclusion into a vaccine. Most antipeptide antisera obtained were reactive with viral protein, and with one of them it was possible to locate the hypothetical amino terminus of VP3 within positions 15-31 of VP2. Virus-neutralizing antibodies were only obtained with two overlapping 15-mer peptides corresponding in sequence to the amino terminus of VP2 (MSDGAVQPDGGQPAVRNERAT). Antibodies in the neutralizing sera bound most strongly to amino acids of the sequence DGGQPAV within the N-terminus of VP2, indicating that efforts to develop a synthetic vaccine against CVP should be focused on this stretch of amino acids. The two peptides induced long-lasting immunity (at least 8 months) using either Freund's adjuvant or aluminium hydroxide plus Quil A. Thus, this approach delineated the exact peptide sequence useful for vaccines applied to the amino-terminal region of VP2. These findings in experimental animals form a solid basis for exploration of a synthetic peptide vaccine against parvovirus infection in dogs, minks or cats.