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.     

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.     

Virus-like particles of recombinant PCV2b carrying FMDV-VP1 epitopes induce both anti-PCV and anti-FMDV antibody responses

Mixed infection of porcine circovirus type 2 (PCV2) and foot-and-mouth disease virus (FMDV) is devastating to swine populations. To develop an effective vaccine that can protect the pigs from the infection of PCV2 and FMDV, we used the neutralizing B cell epitope region (aa 135–160) of FMDV to replace the regions aa 123–151 and aa 169–194 of the PCV2b Cap protein to generate a recombinant protein designated as Capfb. The Capfb protein was expressed in Escherichia coli system and the purified Capfb protein assembled into virus-like particles (VLPs) through dialysis. The ability of the Capfb protein to induce effective immune response against FMDV and PCV2b was tested in mice and guinea pigs. The results showed that the Capfb-VLPs could elicit anti-PCV2b and anti-FMDV antibody response in mice and guinea pigs without inducing antibodies against decoy epitope. Moreover, the Capfb-VLPs could enhance the percentage and activation of B cells in lymph nodes when the mice were stimulated with inactivated FMDV or PCV2b. These data suggested that the Capfb-VLPs could be an efficacious candidate antigen for developing a novel PCV2b-FMDV bivalent vaccine.

     

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。     

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.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.     

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.     

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

Protection against Foot-and-Mouth Disease Virus in Guinea Pigs via Oral Administration of Recombinant Lactobacillus plantarum Expressing VP1

Mucosal vaccination is an effective strategy for generating antigen-specific immune responses against mucosal infections of foot-and-mouth disease virus (FMDV). In this study, Lactobacillus plantarum strains NC8 and WCFS1 were used as oral delivery vehicles containing a pSIP411-VP1 recombinant plasmid to initiate mucosal and systemic immune responses in guinea pigs. Guinea pigs were orally vaccinated (three doses) with NC8-pSIP411, NC8-pSIP411-VP1, WCFS1-pSIP411, WCFS1-pSIP411-VP1 or milk. Animals immunized with NC8-pSIP411-VP1 and WCFS1-pSIP411-VP1 developed high levels of antigen-specific serum IgG, IgA, IgM, mucosal secretory IgA (sIgA) and neutralizing antibodies, and revealed stronger cell-mediated immune responses and enhanced protection against FMDV challenge compared with control groups. The recombinant pSIP411-VP1 effectively improved immunoprotection against FMDV in guinea pigs.     

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.     

Foot-and-mouth disease virus exhibits an altered tropism in the presence of specific immunoglobulins, enabling productive infection and killing of dendritic cells

Foot-and-mouth disease virus (FMDV) causes an acute vesicular disease of farm animals. The development of successful control strategies is limited by an incomplete understanding of the immune response to FMDV. Dendritic cells (DC) mediate the induction of immunity to pathogens, but their role in FMDV infection of cattle is uncharacterized. Bovine monocyte-derived DC (moDC) were exposed to integrin-binding and cell culture-adapted strains of FMDV in vitro. MoDC were not largely susceptible to infection by integrin-binding FMDV but were susceptible to culture-adapted virus. Binding specific antibodies to integrin-binding FMDV at neutralizing or subneutralizing IgG concentrations significantly enhanced infection via CD32 (FcγR). Monocytes also expressed CD32 but were nonsusceptible to FMDV immune complex (IC) infection, indicating a requirement for additional factors involved in cellular susceptibility. Infection of moDC by the FMDV IC was productive and associated with high levels of cell death. Infected moDC were unable to efficiently stimulate FMDV-specific CD4(+) memory T cells, but exposing moDC to IC containing inactivated FMDV resulted in significantly increased T cell stimulation. Thus, neutralized FMDV concurrently loses its ability to infect susceptible cells while gaining the capacity to infect immune cells. This represents a change in the tropism of FMDV that could occur after the onset of the antibody response. We propose that IC could dynamically influence the anti-FMDV immune response and that this may explain why the early immune response to FMDV has evolved toward T cell independence in vivo. Moreover, we propose that DC targeting could prove useful in the development of effective vaccines against FMDV.     

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.     

Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus.

Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.     

Susceptibility of mink to certain viral animal diseases foreign to the United States.

Mink (Mustela vison) were inoculated with viruses: African horse sickness (AHS), African swine fever (ASF), bovine herpes virus II (BHV2), foot-and-mouth disease (FMD), goat pox (GP), hog cholera (HC), peste des petits ruminants (PPR), rinderpest (RP), swine vesicular disease (SVD), vesicular exanthema of swine (VES) and vesicular stomatitis (VS). Their susceptibility was measured by development of clinical signs, virus isolation and detection of precipitin and/or virus neutralizing antibodies. SVD virus produced a lesion in one mink. Virus was isolated from mink inoculated with SVD, FMD and BHV2. Neutralizing and/or precipitin antibodies were detected in mink inoculated with ASF, FMD, GP, RP, SVD and VS viruses. Mink were not susceptible to AHS, HC, PPR and VES viruses.     

A Novel Mucosal Vaccine Against Foot-and-Mouth Disease Virus Induces Protection in Mice and Swine

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6×His-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1–hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1–hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10⁵ FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1–hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10^6.5 FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV. Houhui Song, Zhiliang Wang and Dongxia Zheng contributed equally to this work.     

Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.     

Structures of Foot-and-mouth Disease Virus with neutralizing antibodies derived from recovered natural host reveal a mechanism for cross-serotype neutralization

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.     

Detection of Foot-and-Mouth Disease Virus Antibodies: II. Use of Fractionated Bovine Antisera for Improving the Specificity of a “Passive” Hemagglutination Test1

Because 7S immunoglobulin (Ig) G antibodies of low type specificity were present in mixtures with highly specific 19S IgM antibodies, many bovine antisera to foot-and-mouth disease virus (FMDV) type A(12), strain 119 cross-reacted with type O of FMDV and to some degree with type C in the passive hemagglutination (HA) test. After 19S IgM antibodies were separated by density gradient centrifugation or precipitated with 4% (w/v) polyethylene glycol, the antigen could be determined with "block" HA tests. Such tests used several antigen concentrations in the titration of each antiserum. Adding 4% (w/v) polyethylene glycol to the serum was especially convenient for rapid precipitation of 19S IgM antibodies for the test. Similar results were obtained with bovine 19S IgM antibodies to FMDV type O, subtype 1, strain Caseros and type C strain Rezende.     

一种制备口蹄疫抗原和高免血清的新方法

口蹄疫是一种一类传染病,其病原为口蹄疫病毒(FMDV)。FMDV衣壳蛋白VP1含有中和性抗原表位。本研究合成了编码O型FMDVVP1蛋白中和性抗原表位的双拷贝DNA片段,将其克隆于原核表达载体pGEX-6p-1中,以获得的重组质粒转化大肠杆菌,然后用IPTG进行诱导,表达出了大小约为33kDa的GST融合重组蛋白,Westernblotting分析证实,重组蛋白可以被O型FMDV抗血清所识别。用纯化的重组蛋白免疫豚鼠制备高免血清,ELISA检测表明,免疫后的豚鼠血清抗体滴度可达1∶20560以上。本研究提供了一种制备口蹄疫抗原和高免血清的新方法。