Enhanced mucosal immunoglobulin A response and solid protection against foot-and-mouth disease virus challenge induced by a novel dendrimeric peptide

The successful use of a dendrimeric peptide to protect pigs against challenge with foot-and-mouth disease virus (FMDV), which causes the most devastating animal disease worldwide, is described. Animals were immunized intramuscularly with a peptide containing one copy of a FMDV T-cell epitope and branching out into four copies of a B-cell epitope. The four immunized pigs did not develop significant clinical signs upon FMDV challenge, neither systemic nor mucosal FMDV replication, nor was its transmission to contact control pigs observed. The dendrimeric construction specifically induced high titers of FMDV-neutralizing antibodies and activated FMDV-specific T cells. Interestingly, a potent anti-FMDV immunoglobulin A response (local and systemic) was observed, despite the parenteral administration of the peptide. On the other hand, peptide-immunized animals showed no antibodies specific of FMDV infection, which qualifies the peptide as a potential marker vaccine. Overall, the dendrimeric peptide used elicited an immune response comparable to that found for control FMDV-infected pigs that correlated with a solid protection against FMDV challenge. Dendrimeric designs of this type may hold substantial promise for peptide subunit vaccine development.     

The early protective thymus-independent antibody response to foot-and-mouth disease virus is mediated by splenic CD9+ B lymphocytes

Infection of mice with cytopathic foot-and-mouth disease virus (FMDV) induces a rapid and specific thymus-independent (TI) neutralizing antibody response that promptly clears the virus. Herein, it is shown that FMDV-infected dendritic cells (DCs) directly stimulate splenic innate-like CD9(+) B lymphocytes to rapidly (3 days) produce neutralizing anti-FMDV immunoglobulin M antibodies without T-lymphocyte collaboration. In contrast, neither follicular (CD9(-)) B lymphocytes from the spleen nor B lymphocytes from lymph nodes efficiently respond to stimulation with FMDV-infected DCs. The production of these protective neutralizing antibodies is dependent on DC-derived interleukin-6 (IL-6) and on CD9(+) cell-derived IL-10 secretion. In comparison, DCs loaded with UV-inactivated FMDV are significantly less efficient in directly stimulating B lymphocytes to secrete TI antibodies. A critical role of the spleen in the early production of anti-FMDV antibodies in infected mice was also demonstrated in vivo. Indeed, either splenectomy or functional disruption of the marginal zone of the spleen delays and reduces the magnitude of the TI anti-FMDV antibody response in infected mice. Together, these results indicate that in addition to virus localization, the FMDV-mediated modulation of DC functionality is a key parameter that collaborates in the induction of a rapid and protective TI antibody response against this virus.     

Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.     

Immune potential of a novel multiple-epitope vaccine to FMDV type Asia 1 in guinea pigs and sheep

To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus(FMDV)type Asia 1 in sheep,a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized.The biologically functional molecule,the ovine IgG heavy constant region(oIgG)as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed.The recombinant proteins,RE and RE-oIgG,were expressed as a formation of inclusion bodies in E.coli.The immune potential of this vaccine regime in guinea pigs and sheep was evaluated.The results showed that IgG could significantly enhance the immune potential of antigenic epitopes.The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs,but confer complete protection in guinea pigs against virus challenge.Although the recombinant protein RE could not confer protection in the vaccinated animals,it could delay the appearance of the clinical signs and reduce the severity of disease.Inspiringly,the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination.Therefore,we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.     

Chimeric virus-like particles elicit protective immunity against serotype O foot-and-mouth disease virus in guinea pigs

Foot-and-mouth disease (FMD) is an acute and highly contagious disease caused by foot-and-mouth disease virus (FMDV) that can affect cloven-hoofed animal species, leading to severe economic losses worldwide. Therefore, the development of a safe and effective new vaccine to prevent and control FMD is both urgent and necessary. In this study, we developed a chimeric virus-like particle (VLP) vaccine candidate for serotype O FMDV and evaluated its protective immunity in guinea pigs. Chimeric VLPs were formed by the antigenic structural protein VP1 from serotype O and segments of the viral capsid proteins (VP2, VP3, and VP4) from serotype A. The chimeric VLPs elicited significant humoral and cellular immune responses with a higher level of anti-FMDV antibodies and cytokines than the control group. Furthermore, four of the five guinea pigs vaccinated with the chimeric VLPs were completely protected against challenge with 100 50% guinea pig infectious doses (GPID₅₀) of the virulent FMDV strain O/MAY98. These data suggest that chimeric VLPs are potential candidates for the development of new vaccines against FMDV.     

重组鸡痘病毒和DNA疫苗对口蹄疫病毒的豚鼠免疫保护

将构建的携带FMDV衣壳蛋白P1-2A和蛋白酶3C编码基因的重组鸡痘病毒活载体疫苗vUTAL3CP1以及编码FMDVP1-2A基因和猪IL-18基因的重组DNA疫苗pVIRIL18P1,分别以单独和混合的方式给豚鼠进行2次免疫,然后测定FMDV特异性结合抗体、中和抗体和T淋巴细胞增殖反应,并用250ID50的FMDV进行攻击,观察其保护效果。结果表明这2种基因工程疫苗均能诱导豚鼠产生特异性的体液免疫及细胞免疫应答。其中以vUTAL3CP1两次免疫组的效果最好,其诱导的抗体水平已接近于常规灭活疫苗,而细胞免疫水平则比后者高得多。攻击保护结果表明该组完全保护率可达3/4,而另外两组也具有一定保护效果。上述研究结果为进一步进行大动物免疫攻毒试验,并最终筛选出最佳疫苗和免疫程序奠定了基础。     

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.     

Immunogenicity of Two FMDV Nonameric Peptides Encapsulated in Liposomes in Mice and the Protective Efficacy in Guinea Pigs

It has been predicted that nonameric peptides I (VP1_26–34, RRQHTDVSF), II (VP1_157–165, RTLPTSFNY) and III (VP1_45–53, KEQVNVLDL) from the VP1 capsid protein of the foot-and-mouth disease virus (FMDV) are T cell epitopes. To investigate whether these peptides have immunological activity, BALB/c mice were immunized with peptide I, II or III conjugated with immunostimulating complexes (ISCOMs). A cytotoxic T lymphocyte assay was used to evaluate the cytotoxic activity induced by peptides along with by measuring peptide-specific T-cell proliferation and CD8⁺ T lymphocyte numbers in whole blood and interferon (IFN)-γ production in peripheral blood mononuclear cells induced by peptides. To further identify the protective efficacy of peptides, an FMDV challenge assay was done in guinea pigs. Peptides I and II stimulated significant increases in T-cell proliferation, CD8⁺ T lymphocytes, and IFN-γ secretion and cytotoxic activity compared to controls. The FMDV challenge assay indicated peptides I and II can protect over 60% of animals from virus attack. The results demonstrate that peptides I and II encapsulated in liposomes should be CTL epitopes of FMDV and can protect animals from virus attack to some extent.     

Molecular dynamics of the alpha-helical epitope of a novel synthetic lipopeptide foot-and-mouth disease virus vaccine

A novel synthetic foot-and-mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B-cell and macrophage activator covalently linked to an amphiphilic alpha-helical T-cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P3CSS) as built-in adjuvant. The vaccine, tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl-FMDV-VP1 (VP1 = serotype O1K 135-154) induces protection against homologous challenge and serotype-specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV-VP1 segment 135-154 of strain O Wuppertal produced only poor cross-protection against challenge with O1K virus. The antigenic determinant VP1(135-154) is an amphiphilic alpha-helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous alpha-helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138-149) suggest that the FMDV segment 138-149 may adopt alpha-helical conformation during binding to its T-cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.     

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.     

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

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

Recombinant plasmids containing hybrid protein genes with antigenic determinants of the foot and mouth virus

Plasmids have been constructed which contain genes coding for fused proteins including beta-galactosidase or human leukocyte interferon alpha 2 and monomeric or pentameric form of the main antigenic determinant of the foot-and-mouth disease virus (FMDV) serotype 01K. Expression of the hybrid genes has been studied. It is shown that fused proteins, containing beta-galactosidase and the antigenic determinant (monomer or pentamer), interact specifically with anti-FMDV anti-sera and with antibodies against peptide 141-160 of FMDV VP1 coat protein.     

A Combinatorial Peptide Library Around Variation of the Human Immunode ficiency Virus (HIV-1) V3 Domain Leads to Distinct T Helper Cell Responses

The hypervariable domain of the HIV gp120, the V3 loop domain, represents a target for neutralizing antibodies and for HIV vaccine strategies. In this study, we have investigated in murine species the potential cross-reactivity of immune responses elicited by immunization either with individual V3 peptides, derived from distinct HIV sequences (BRU, RF, SF2, MN and ELI sequences), or with a V3 combinatorial peptide library. We observed that individual V3 peptides are immunogenic but elicit a specific B- and T-cell immune response that is mainly restricted to the sequence of the immunizing peptide. In particular, T-cell responses that depend on T-cell receptor recognition of peptides bound to the molecules encoded by the major histocompatibility complex were significantly influenced by small differences in the peptide amino acid sequence. The combinatorial V3 peptide library, previously described as B- and T-cell immunogens, induced a more broadly reactive immune response, specially when T-cell cytokine secretion was used as a readout for restimulation of T-cells with individual V3 peptides. These data suggest that amino acid variations in the sequence of an antigenic peptide could lead to the induction of different transducing signals in the primed T-cell population and to the activation of T-cells with distinct cytokine secretion properties. These observations may have implications in the understanding of antigenic variability and in the design of vaccine strategies.     

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.     

一种实用的筛选病毒抗原表位方法的建立(英)

采用基因分段克隆、表达结合蛋白质印迹, 筛选到了口蹄疫病毒非结构蛋白3ABC上高结合力、保守的感染相关线性表位,分别位于3ABC蛋白上第106～155和156～190位氨基酸.这两个表位可与感染不同血清型口蹄疫病毒动物康复血清反应,但不与来自健康免疫动物和未接触病毒动物的血清发生反应.实验表明,用基因工程表达的多肽筛选抗原表位的方法是可行的.     

Use of substituted and tandem-repeated peptides to probe the relevance of the highly conserved RGD tripeptide in the immune response against foot-and-mouth disease virus

Antigenic site A of foot-and-mouth disease virus (FMDV) is an exposed, mobile loop which includes a central, highly conserved Arg-Gly-Asp tripeptide (RGD, VP1 residues 141–143 in serotype C) thought to be part of the cell attachment site. We have analyzed the contribution of RGD to the interaction of site A with antibodies by incorporating selected amino acid replacements at RGD into synthetic peptides representing site A, and analyzing the reactivity of substituted peptides with site A-specific monoclonal antibodies (MAbs). Replacement of Arg-141, Gly-142 or Asp-143 by alanine resulted in the loss of one, three and five epitopes, respectively, out of seven epitopes probed. Other replacements resulted in the loss of even larger numbers of epitopes, suggesting that the amino acids of the RGD region are either directly involved in interaction with antibodies or that they exert an important influence on the interaction of surrounding residues with antibodies. Thus, we explored the ability of tandem repeats of the RGDL sequence (corresponding to FMDV C-S8cl) to evoke neutralizing antibodies in rabbits and guinea pigs. Neutralizing activity was generally low but with a broad specificity for different FMDV serotypes and variants. Significant decreases in neutralizing titers were observed with boosting, suggesting a possible suppression of those anti-peptide antibodies which may also be directed to cellular RGD sequences. The results point to an involvement of RGD in the antigenic structure of site A, and open the possibility that broadly neutralizing antibodies might be induced by tandem repeats of the critical, conserved domain.     

An ELISA based on the repeated foot-and-mouth disease virus 3B epitope peptide can distinguish infected and vaccinated cattle

To develop a strategy of differentiating infected from vaccinated animals (DIVA) with foot-and-mouth disease virus (FMDV), a short (27aa) peptide containing three conserved linear B cell epitopes of the FMDV 3B nonstructural protein was designed. This novel BF peptide was synthesized using a gene splicing by overlap extension protocol with preferred codons for Escherichia coli. The resultant eight tandem repeat multimer (1, 2, 4, 6, 8, 16, 24, and 32BF) were expressed as soluble fusion proteins in E. coli. An indirect ELISA was developed based on the recombinant 8BF protein with the aim of specifically distinguishing antibodies induced by FMDV infection but not those induced by vaccination. Using the cut-off value of 0.3, the sensitivity of the assay was 96.8% and the specificities for naive and vaccinated cattle were 99.8 and 99.0%, respectively. The performance of the newly developed epitope-based ELISA was compared with three commercial NSP ELISA kits. The 8BF-ELISA appears to be a promising DIVA test for FMD control and eradication.     

Fimbriae of Bacteroides nodosus: protein engineering of the structural subunit for the production of an exogenous peptide

The pattern of sequence variation between Bacteroides nodosus fimbrial subunits of different serotypes suggests a degree of flexibility, which might be exploited for protein engineering approaches for the expression of other peptides. We have tested this using the well-characterized peptide epitope from VP1 of foot-and-mouth disease virus (FMDV), residues 144-159: LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage, several oligonucleotides were designed for the substitution of this sequence internally at hypervariable regions of the fimbrial subunit (aligned for maximum homology), and for its addition at the carboxyterminus with a diglycine spacer as a flexible hinge. Following site-directed mutagenesis in phage M13, the modified genes were placed under PL promoter control and placed in a broad host range vector. Analysis of the variant proteins expressed in E. coli showed that these substitutions affected, to varying extents, recognition by both anti-fimbrial and anti-FMDV antibodies, indicating that hypervariable region 2 is a major antigenic determinant of the fimbrial subunit and that local stereochemical effects can influence antibody binding to the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa, viable transformants could only be obtained with the mutant gene encoding the carboxy-terminal graft. These cells provided fimbrial preparations comprised of the modified subunit. This then constitutes the prototype for the development of a general expression system for the production of vaccine epitopes and other bioactive peptides. Furthermore, there is considerable scope for further modification of the system, for example by engineering specific chemical or protease cleavage sites for release of the grafted peptide. Alternatively, the fimbriae themselves may serve as a useful supramolecular carrier or adjuvant for immune provocation.