Homologous Foot-and-mouth Disease Virus Research Articles

Preparation of a polysaccharide adjuvant and its application in the production of a foot-and-mouth disease virus-like particles vaccine

Foot-and-mouth disease (FMD) virus-like particles (VLPs) comprise a new and effective FMD vaccine, which often requires the addition of adjuvants to induce a vigorous immune response in order to protect livestock from the challenge of FMD virus (FMDV). Oil emulsion delivery systems have been developed and used widely in the field of veterinary vaccines because of their low cost, slow release, and efficient antigen protection. In this study, we designed and synthesized an optimized Achyranthes bidentata polysaccharide (ABP) delivery system (ABPOA) based on a pseudo-ternary phase diagram. We systematically evaluated the stability, biocompatibility, and immunological effects of the ABP delivery system. The results demonstrated that ABPOA is a stable adjuvant delivery system and capable of long-term storage at 37 °C for up to 6 months. ABP-VLPs can be effectively stored for long periods when ABPOA is used as the delivery system for VLPs. Importantly, guinea pig-specific antibodies were significantly increased within 28 days when ABPOA was used as a delivery system, thereby promoting the proliferation of splenic lymphocytes. In addition, immunization with one dose of ABP-VLPs resulted in complete protection from homologous FMDV challenge in guinea pigs. Therefore, the polysaccharide oil emulsion adjuvant comprising ABPOA effectively improved the immunogenicity of FMD-VLPs. These findings provide a theoretical basis for the preparation of oil emulsion adjuvants for veterinary vaccines.

Needleless intradermal vaccination for foot-and-mouth disease induced granuloma-free effective protection in pigs.

Vaccination is one of the most effective ways of controlling and preventing foot-and-mouth disease (FMD) outbreaks. The effective prevention of this disease requires the use of high-quality vaccines to meet the criteria that enable customers to use them simply. The administration of FMD vaccines containing oil-based adjuvants in pigs can induce the formation of granuloma in the muscle of the vaccinated, which makes these vaccines a less preferable option. Therefore, it is important to establish an FMD vaccine and vaccine delivery tool that offers better immunity and safer application. This study compared the immune responses of intramuscular and needleless intradermal vaccination in pigs. When the same amount of an FMD virus (FMDV) antigen was administered to pigs, both the intradermally and intramuscularly vaccinated groups were protected completely against a challenge of the homologous FMDV, but the intramuscularly vaccinated group showed an overall higher level of neutralizing antibodies. Importantly, the formation of granuloma in muscle could be excluded in the intradermally vaccinated group. Of the oil-based adjuvants selected in this study, ISA 207 was effective in eliciting immunogenicity in intradermal vaccination. In conclusion, a new vaccine formula can be chosen for the delivery of intradermal route to exclude the possibility of local reactions in the muscle and generate protective immunity against an FMDV challenge.

Chemokine CCL20 plasmid improves protective efficacy of the Montanide ISA™ 206 adjuvanted foot-and-mouth disease vaccine in mice model

This study aimed to investigate the chemokine CCL20, a macrophage inflammatory protein-3 alpha, for adjuvant potential in inactivated foot-and-mouth disease (FMD) vaccine. Groups of mice were injected intramuscularly with either murine CCL20 DNA or CCL20 protein two days ahead of the immunization with Montanide ISA206 adjuvanted inactivated FMD vaccine and humoral and cellular immune responses were measured in post-vaccinal sera. We demonstrated that the mice immunized with CCL20 plasmid plus FMD vaccine showed earlier and significantly (p < 0.05) higher neutralizing antibody responses compared to the mice vaccinated with CCL20 protein plus FMD vaccine. In fact, CCL20 as a protein did not show any adjuvant effect and the immune responses induced in this group were comparable to that of the mice vaccinated with FMD vaccine alone. All the vaccination groups showed serum IgG1 and IgG2 antibody responses; however, the mice vaccinated with CCL20 plasmid plus FMD vaccine showed significantly (p < 0.05) higher IgG1 and IgG2 responses and the responses remained high at all-time points post vaccination, although not always statistically significant. Upon restimulation of the vaccinated splenocytes with the inactivated FMD viral antigen, significantly (p < 0.05) higher IFN-γ and IL-2 levels in culture supernatants were found in animals vaccinated with the CCL20 plasmid plus FMD vaccine, which is indicative of the TH1 type of cellular immunity. On challenge with the homologous FMD virus on 28th day post immunization, CCL20 plasmid plus FMD vaccine showed complete protection (100%) while animals immunized with CCL20 protein plus FMD vaccine or FMD vaccine alone showed 66% protection. In summary, we show that prior injection of CCL20 plasmid improved protective efficacy of the inactivated FMD vaccine and thus offers a valuable strategy to modulate the efficacy and polarization of specific immunity against inactivated vaccines.

Coinjection of a vaccine and anti-viral agents can provide fast-acting protection from foot-and-mouth disease

Foot-and-mouth disease (FMD) is the cause of an economically devastating animal disease. With commercial inactivated FMD vaccines, the protection against FMD virus (FMDV) begins a minimum of 4 days post vaccination (dpv). Therefore, antiviral agents could be proposed for rapid protection and to reduce the spread of FMDV during outbreaks until vaccine-induced protective immunity occurs. In previous studies, we have developed two recombinant adenoviruses that simultaneously express porcine interferon-α and interferon-γ (Ad-porcine IFN-αγ) and multiple siRNAs that target the non-structural protein-regions of FMDV (Ad-3siRNA), and we have shown that the combination of the two antiviral agents (referred to here as Ad combination) induced robust protection against FMDV in pigs. In an attempt to provide complete protection against FMDV, we co-administered Ad combination and the FMD vaccine to mice and pigs. In the C57BL/6 mice model, we observed rapid and continuous protection against homologous FMDV challenge from 1 to 3 dpv—the period in which vaccine-mediated immunity is absent. In the pig experiments, we found that most of the pigs (five out of six) that received vaccine + Ad combination and were challenged with FMDV at 1 or 2 dpv were clinically protected from FMDV. In addition, most of the pigs that received vaccine + Ad combination and all pigs inoculated with the vaccine only were clinically protected from an FMDV challenge at 7 dpv. We believe that the antiviral agent ensures early protection from FMDV, and the vaccine participates in protection after 7 dpv. Therefore, we can say that the combination of the FMD vaccine and effective antiviral agents may offer both fast-acting and continuous protection against FMDV. In further studies, we plan to design coadministration of Ad combination and novel vaccines.

The B Cell Response to Foot-and-Mouth Disease Virus in Cattle following Sequential Vaccination with Multiple Serotypes.

Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype does not confer interserotype protection. However, some historical data have shown that interserotype protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study, we have investigated the kinetics of the FMDV-specific antibody-secreting cell (ASC) response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar for all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O, A, and Asia1 serotypes) a B cell response to FMDV SAT1 and serotype C was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific ASCs in the absence of circulating FMDV-specific ASCs, indicating the presence of short-lived ASCs, a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid.IMPORTANCE We have demonstrated the development of intraserotype response following a sequential vaccination regime of four different FMDV serotypes. We have found indication of short-lived ASCs in the local lymphoid tissue, further evidence of a TI-2 response to FMDV.

Adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against FMDV O1 Manisa in swine

A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 107 pfu/animal while a dose of 4×107pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine.

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge

The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×108 to 2.1×1011 particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R2=0.97) and viremia (R2=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R2=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.

Intra-serotype SAT2 chimeric foot-and-mouth disease vaccine protects cattle against FMDV challenge

The genetic diversity of the three Southern African Territories (SAT) types of foot-and-mouth disease virus (FMDV) reflects high antigenic variation, and indications are that vaccines targeting each SAT-specific topotype may be needed. This has serious implications for control of FMD using vaccines as well as the choice of strains to include in regional antigen banks. Here, we investigated an intra-serotype chimeric virus, vSAT2ZIM14-SAT2, which was engineered by replacing the surface-exposed capsid-coding region (1B-1D/2A) of a SAT2 genome-length clone, pSAT2, with that of the field isolate, SAT2/ZIM/14/90. The chimeric FMDV produced by this technique was viable, grew to high titres and stably maintained the 1B-1D/2A sequence upon passage. Chemically inactivated, oil adjuvanted vaccines of both the chimeric and parental immunogens were used to vaccinate cattle. The serological response to vaccination showed the production of strong neutralizing antibody titres that correlated with protection against homologous FMDV challenge. We also predicted a good likelihood that cattle vaccinated with an intra-serotype chimeric vaccine would be protected against challenge with viruses that caused recent outbreaks in southern Africa. These results provide support that chimeric vaccines containing the external capsid of field isolates induce protective immune responses in FMD host species similar to the parental vaccine.

Immunity of Foot-and-Mouth Disease Serotype Asia 1 by Sublingual Vaccination

Foot-and-mouth disease virus (FMDV) causes vesicular disease of cloven-hoofed animals, with severe agricultural and economic losses. Here we present study using a sublingual (SL) route with the killed serotype Asia 1 FMDV vaccine. Guinea pigs were vaccinated using a commercially available vaccine formulation at the manufacturer’s recommended full, 1/4, and 1/16 antigen doses. Animals were challenged with homologous FMDV Asia1 strain at various times following vaccination. All control guinea pigs exhibited clinical disease, including fever, viremia, and lesions, specifically vesicle formation in feet. Animals vaccinated with the 1/16 and 1/4 doses were protected after challenge at days 7, 28, and 35 post vaccination. These data suggest that effective protection against foot-and-mouth disease can be achieved with 1/16 of the recommended vaccine dose using SL vaccination, indicating that the sublingual route is an attractive alternative for the administration of the FMDV vaccine.

An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry.

AttenuatedSalmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 109 CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID50 of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 109 CFU of p3D-NT56/S. cho were protected in 9 days.

Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease

Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1–3 × 10 8 TCID 50 Ad-P12 × 3C recombinant adenovirus were completely protected against 10,000 GID 50 homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12 × 3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1 × 10 9 TCID 50 Ad-P12 × 3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID 50) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12 × 3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

Cedivac-FMD can be used according to a marker vaccine principle

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest ® FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine . All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest ® FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD 50 vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7–14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of ‘marker vaccine’ applies.

Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody

A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector antibody. Sera from naive, vaccinated and infected cattle, sheep and pigs were examined. The specificity of the test was high. Non-specific reactions observed in particular in sera of cattle and sheep could be removed by filtration and inactivation. Positive reactions were obtained for sera from cattle infected with all seven serotypes of FMDV. The test detected antibodies from days 7 or 9 following experimental infection of non-vaccinated cattle and sheep, and in cattle strong positive reactions persisted for up to 395 days after infection. In vaccinated cattle that became carriers after challenge with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISA's when used to test sera from cattle, pigs and sheep collected after experimental or natural infection. The blocking ELISA based on recombinant FMDV 3ABC antigen and a monoclonal antibody to 3ABC is a promising tool for FMD control and eradication campaigns, where vaccination has been carried out.

Immediate protection of swine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine

We have previously shown that swine inoculated with recombinant, replication-defective human adenovirus type 5 containing the porcine interferon alpha gene (Ad5-pIFNα) are completely protected when challenged 1 day later with virulent foot-and-mouth disease virus (FMDV). In the current study, we examined the duration of protection afforded swine by Ad5-pIFNα and the ability of a combination of Ad5-pIFNα and a FMDV subunit vaccine delivered by Ad5-A24 (an Ad5 vector containing the capsid coding region of FMDV serotype A24 Cruzeiro and the 3C proteinase coding region of FMDV serotype A12) to induce immediate as well as long-lasting protection against homologous FMDV challenge. Groups of swine were inoculated with Ad5-pIFNα and challenged with virulent FMDV A24 1, 3, 5, and 7 days postinoculation (dpi) or 1 day preinoculation. All animals challenged 1 and 3 dpi were completely protected from disease. The animals in the remaining groups had either no clinical signs of disease or clinical signs were delayed and less severe compared to the control group. Swine inoculated with a combination of Ad5-pIFNα and Ad5-A24 and challenged 5 dpi were all completely protected from disease and developed a significant FMDV-specific neutralizing antibody response.

Stable expression of antisense RNAs targeted to the 5' non-coding region confers heterotypic inhibition to foot-and-mouth disease virus infection.

The antiviral potential of transcripts targeted to the non-coding regions (NCRs) of foot-and-mouth disease virus (FMDV) RNA have been studied during transient and constitutive expression in susceptible BHK-21 cells. Transient expression of antisense transcripts corresponding to the 5' and 3'NCRs, alone or in combination, confers specific inhibition of homologous (serotype C) virus infection in BHK-21 cells. Constitutive expression of antisense 5'NCR transcripts (5'AS) exerted higher levels of inhibition to homologous and heterologous (serotypes O, A, Asia, SAT 1, SAT 2 and SAT 3) FMDV infection, as estimated by a 10-fold reduction in virus titre in the supernatants from infected clones and by a plaque reduction assay. These inhibitions were also observed, albeit to a lesser extent, in clones stably expressing antisense 3'NCR transcripts. The antiviral response was specific for FMDV, as the picornavirus encephalomyocarditis virus was not inhibited in any of the transformed cell lines. In all cases, a correlation was found between the level of transcript expression and the extent of virus inhibition. The potential to efficiently inhibit FMDV, including isolates representing the seven serotypes, by expressing interfering 5'AS transcripts opens the possibility of developing transgenic animals with a reduced susceptibility to FMDV.

A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot-and-mouth disease types A, O and C II. Application

The complex-trapping-blocking (CTB) enzyme-linked immunosorbent assay (ELISA) was evaluated to detect antibodies directed against foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, and C1 Detmold. Log 10 serum titres of uninfected, unvaccinated cattle ( n = 100) were less than 1.80 in the CTB-ELISA. Sera from cattle vaccinated with either monovalent or trivalent vaccines were tested in both the CTB-ELISA and the serum neutralisation test (SNT); titres in both tests correlated positively ( P < 0.001). Titres of sera from cattle, sheep, and pigs vaccinated twice with FMDV A10 Holland also correlated positively in both tests. In another experiment, cattle vaccinated with FMDV strain C1 Detmold were intradermolingually challenged 3 weeks after primary vaccination; at the same time two controls were challenged. At 8 days after challenge, serum titres of the controls were distinctly higher in the CTB-ELISA than in the SNT, whereas serum titres of the vaccinated cattle were equally high in both tests. In potency tests for monovalent vaccines against FMDV strains A10 Holland, O1 BFS or C1 Detmold, serum titres correlated strongly in both tests with protection against the homologous FMDV strain. We concluded that the CTB-ELISA is not only sensitive, but easier to perform and more rapid and reproducible than the SNT. The CTB-ELISA may be useful in evaluating the immune response in cattle during FMD vaccine potency tests.

Analysis of Foot-and-Mouth Disease Virus Type O1 Brugge Neutralization Epitopes Using Monoclonal Antibodies

Monoclonal antibodies (MAbs) were elicited with inactivated, purified foot-and-mouth disease virus (FMDV) type O1 strain Brugge (140S) and with 12S protein subunits. Each MAb was tested for its capacity to bind to FMDV O1 Brugge 140S virions, 12S subunits and purified VP1 by radioimmunoassay (RIA) and to neutralize viral infectivity in mouse protection assays. Those MAbs which reacted only with 12S subunits in RIA did not neutralize infectious virus. One MAb, 12FE9.2.1, reacted with 140S, 12S and purified VP1 of FMDV O1 Brugge and neutralized infectious virus. Reactions with different biosynthetic FMDV type O1 Campos VP1 polypeptides localized the binding site of 12FE9.2.1 between amino acid residues 135 and 172. Eight MAbs reacted with both 140S virions and 12S subunits and neutralized infectious virus. Monoclonal antibodies that reacted only with 12S protein subunits or with 140S, 12S and VP1 did not compete in RIA with 140S/12S-reactive neutralizing MAbs for 12S binding sites. The ability of 140S/12S-reactive MAbs to compete for the 12S binding site defined by MAb 10GA4.2.2 was directly related to their capacity to neutralize infectious virus, suggesting that these MAbs were all affecting a single neutralization site. In mouse protection and plaque reduction neutralization assays, 10GA4.2.2 and 12FE9.2.1 were equally effective in neutralizing the homologous FMDV type O1 Brugge, and in cross-neutralization assays both exhibited high titres against four additional strains of type O1 FMDV.

Location and characterization of the antigenic portion of the FMDV immunizing protein.

Purified foot-and-mouth disease virus (FMDV) to type O1K was treated with several endopeptidases of differing specificity. The immunizing protein VPThr was cleaved into two detectable fragments by all enzymes except for glutamic acid-specific Staphylococcus aureus V8 protease. The longest fragments were generated by mouse submaxillary gland protease and the shortest by trypsin treatment of the intact virion. Several fragments, including the peptides resulting from the cyanogen bromide (CNBr) cleavage of the isolated protein VPThr were characterized in terms of their molecular weights N- and C-terminal amino acids, and ability to induce virus-specific antibodies. The order of the fragments along the protein was determined, and then located on the amino acid sequence of the protein. Two enzyme-sensitive areas of the protein were found on the surface of the virion: between sequence positions 138 and 154 and between portion 200 and the C terminus. Peptides containing these sections were able to induce neutralizing antibodies against the homologous FMDV. When the virus was treated with trypsin or with chymotrypsin, several amino acids between the detectable fragments were lost and the infectivity of the virus was reduced. The infectivity was retained, however, when the enzyme treatment resulted in cleavage of protein with no loss of amino acids or only the cutting away of the C-terminal section. These results suggest that the property of cell attachment is restricted to small regions of the surface of the virus particle.