VP1 Antigen Research Articles

Purification and characterization of the major group-specific core antigen VP7 of bluetongue virus synthesized by a recombinant baculovirus

The major core protein, VP7, of bluetongue virus serotype 10 (BTV-10) has been purified from insect cells infected with a genetically manipulated recombinant baculovirus. The high level expression of VP7 (in excess of 100 mg per litre of culture) and its presence in the soluble fraction of infected cells following lysis by detergent has allowed the purification of the protein virtually to homogeneity (95%) by a simple two-step procedure of ammonium sulphate fractionation and ion-exchange chromatography. The purified antigen is highly immunogenic and has been shown in an ELISA to be reactive with antisera of 24 BTV serotypes (1 to 24) as well as with an antiserum raised to African horsesickness virus type 4 (AHSV-4), a representative of another serogroup of orbiviruses. In confirmation of these data a monospecific antiserum raised with the expressed product has been shown by Western blot analyses to react with other BTV serotypes as well as with two serotypes of epizootic haemorrhagic disease virus (EHDV-1 and EHDV-2), a closely related orbivirus. The data indicated that VP7 is a highly conserved protein amongst BTV serotypes and at least partly conserved amongst three serogroups of orbiviruses.

Expression of rotavirus VP7 antigens in fusions with bacterial proteins

We have cloned antigenic regions of the VP7 gene from rotavirus RV-5 into the lamB gene. The insertions discussed in this paper comprise 250 bp of rotavirus DNA and cover the A and B antigenic regions of the protein. The fusion proteins are expressed and are present in the outer membrane: they react with anti-rotavirus antibody. However, as yet we have not been able to demonstrate that the fusion has LamB protein functions with regard to maltodextrins or L phage, and the fusion protein appears to exist in the outer membrane as a monomer rather than as a trimer.

Antigenic parvovims B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system

Two baculovirus expression vectors derived from Autographica californica nuclear polyhedrosis virus (AcNPV) were prepared containing the complete 2.5 kb coding region for parvovirus B19 coat protein VP1 (AcB19VP1L) and the 1.8 kb coding region for VP2 (AcB19VP2L), placed under the control of the polyhedrin promoter. The recombinant viruses were used to infect Spodoptera frugiperda cells and the proteins expressed were analysed using appropriate antibodies. AcB19VP1L-infected cells produced B19 VP1 as shown by its reaction with 13 human sera containing B19-specific antibodies in Western blot analysis and indirect immunofluorescence. The signal seen with VP1 in immunofluorescence makes it suitable for the development of a diagnostic assay based on this technique. VP1 also reacted with two monoclonal antibodies (mAbs) specific for the B19 protein part of a 196 kDa beta-galactosidase B19 fusion protein expressed in E. coli. Cells infected with AcB19VP2L produced B19 VP2 which reacted with the same human sera in indirect immunofluorescence and with five of the 13 sera in Western blots. VP2 did not react with the fusion protein-specific mAbs. The large amounts of viral antigen produced in this system means the development of widely available diagnostic tests for B19 infection and the further characterization of the B19 structural proteins are within reach.

Lack of cross-reaction between myelin basic proteins and putative demyelinating virus envelope proteins

No cross-reaction could be detected between purified myelin basic proteins (MBP) from mouse, rat or human origins and envelope proteins of viruses suspected of inducing demyelinating processes. In the experimental model using Theiler's murine encephalomyelitis virus, competition radioimmunoassay failed to detect any cross-reaction between MBP and VP1, VP2 and VP3 envelope antigens. In the human situation, antibodies against SV5 and measles viruses, both etiologically linked with multiple sclerosis, also failed to recognize MBP. These results rule out molecular mimicry as a cause of demyelination.

CHRONIC ENTEROVIRUS INFECTION IN PATIENTS WITH POSTVIRAL FATIGUE SYNDROME

76 patients with the postviral fatigue syndrome (PVFS) and 30 matched controls were investigated. Virus isolation was attempted from concentrated faecal samples by direct culture and after acid dissociation of virus from antibody. Positive cultures of enteroviruses were obtained from 17 (22%) patients and 2 (7%) controls. An enterovirus-group-specific monoclonal antibody, 5-D8/1, directed against the VP1 polypeptide, was used to detect enteroviral antigen in the circulation, either free or complexed with antibody. VP1 antigen was detected in the serum of 44 (51%) of a further group of 87 PVFS patients. The number of patients positive for VP1 antigen was greater (42/44) when IgM complexes were detectable than when they were not (2/23). 1 year later, the 17 patients of the first group of 76 with positive cultures were again studied. The same virus was again isolated from 5 (29%), 13 (76%) had detectable IgM responses to enteroviruses, and 9 (53%) were positive for VP1 antigen in the serum. These results show that chronic infection with enteroviruses occurs in many PVFS patients and that detection of enterovirus antigen in the serum is a sensitive and satisfactory method for investigating infection in these patients.

Cis- and trans-acting sequences required for expression of simian virus 40 genes in mouse oocytes.

To determine the requirements for gene expression in mammalian germ cells, circular double-stranded simian virus 40 (SV40) DNA molecules containing deletions in sequences controlling transcription and replication were injected into the nucleus of mouse oocytes. Expression of large (T-Ag) and small (t-Ag) tumor antigens ("early gene products") required at least three GGGCGG boxes, but did not require either the origin of viral DNA replication (ori) or a TATA box. Expression of capsid antigen VP1 ("late gene products") required at least three GGGCGG boxes, sequences between nucleotides 197 and 273 in the 72-bp repeat region, and transactivation by T-Ag. These results are consistent with the requirements for expression of the same genes in differentiated mammalian cells. Surprisingly, however, the 72-bp repeats ("enhancer elements") that are required for expression of T-Ag and t-Ag genes in differentiated cells were not required in mouse oocytes. Similarly, expression of both the early and late genes was unaffected in mouse oocytes by the absence of either DNA replication or an intact ori sequence, components required for maximum expression of late genes in differentiated cells. Thus, mammalian oocytes effectively utilize promoters that are fully active in mammalian differentiated cells only when associated with either enhancer elements or DNA replication. Furthermore, requirements for expression of SV40 genes in mouse oocytes are distinctly different from those reported for Xenopus oocytes. This suggests that caution should be exercised when extrapolating conclusions drawn from experiments with amphibian germ cells to mammalian germ cells.

Expression of bovine rotavirus neutralization antigen in Escherichia coli.

A 646 bp fragment derived from a full length cDNA clone of genomic segment 9 of bovine rotavirus (NCDV strain) was inserted into Escherichia coli expression plasmid pEX1. The fragment encodes amino acids 50 to 265 of the major vital neutralization antigen VP7, a 326 amino acid long outer shell glycoprotein. Several transformed bacterial clones were isolated in which the recombinant plasmid directed the synthesis of a cro-beta-galactosidase-VP7 fusion protein that was recognized by rabbit polyclonal antibodies against NCDV rotavirus. Sera from rabbits immunized with the fusion protein specifically reacted with VP7 among NCDV virion polypeptides. The chimeric polypeptide was also specifically recognized by two monoclonal antibodies against UK strain rotavirus VP7 that exhibited virus-neutralizing activity. However, immune sera to the chimeric polypeptide showed no neutralizing activity against bovine rotavirus. These results are discussed in view of a recent report that a fusion VP7-beta-galactosidase polypeptide comprising 35 more amino acids at the carboxy terminus was able to induce neutralizing antibodies in mice to simian rotavirus SA11.

Expression of hepatitis A virus cDNA in Escherichia coli: antigenic VP1 recombinant protein.

The genome of hepatitis A virus (HAV) was reverse transcribed into cDNA and molecularly cloned. cDNA clones coding for the capsid protein VP1 that carries the major HAV antigen were cloned into the expression vector pUR290 and expressed in Escherichia coli. The recombinant fusion protein reacted in an immunoblot with rabbit anti-HAV serum, suggesting that it possesses HAV antigenicity.

Production and characterization of the neutralization antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector

DNA representing RNA segment 2 of bluetongue virus (BTV) serotype 10, corresponding to the gene that codes for the BTV neutralization antigen VP2, has been inserted into a baculovirus transfer vector in lieu of the 5′ coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After cotransfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived recombinant transfer vector DNA, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with one of these recombinant viruses, a protein that was similar in size and antigenic properties to the BTV VP2 protein was synthesized. Antibodies raised in mice or rabbits to the baculovirus expressed VP2 protein neutralized the infectivity of BTV-10 virus and to lesser extents BTV serotype 11 and 17 viruses but not BTV-13 virus.

Genetic Reassortment between Two Human Rotaviruses Having Different Serotype and Subgroup Specificities

Two reassortant viruses were selected from a mixed infection of MA104 cells with human rotavirus strains Wa (serotype 1-subgroup II) and HN126 (serotype 2-subgroup I). Antigenic characterization and genotype analysis by polyacrylamide gel electrophoresis revealed that they were reassortants with novel antigenic compositions, i.e. serotype 1-subgroup I (C116) and serotype 2-subgroup II (C15). Furthermore one of them, C15, was considered to have a mosaic antigenicity defined by two serotype-specific antigens, namely the serotype 1-specific VP3 antigen and the serotype 2-specific VP7 antigen. Although this reassortant was shown to be a serotype 2 virus on the basis of its preferential reactivity in neutralization reactions with serotype 2 antiserum, unexpectedly the antiserum prepared against C15 equally neutralized both serotype 1 and serotype 2 strains.

Biotechnological approach to a new foot-and-mouth disease virus vaccine.

Major contributions towards the development of an absolutely safe FMDV vaccine are evident. With the identification of VP1 as the immunogenic protein, it is possible to manufacture a subunit vaccine via biotechnology. DNA sequences encoding the VP1 protein can be introduced into a bacterium with ease; under the appropriate conditions, large amounts of VP1 can be produced in a short time. The accumulation of amino acid sequences generated by recombinant DNA techniques allows identification of antigenic domains, which are the basis of variability among serotype and subtype viruses. As a result, vaccine production by chemical synthesis of short peptides corresponding to the antigenic determinants is greatly facilitated. At present, results from experimental vaccines employing genetically engineered or chemically synthesized VP1 antigens against homologous virus infection are encouraging. The current approach of preparing vaccine is to utilize the antigenic specificity of the virus. Since FMDV undergoes antigenic drift, variants not neutralized by type-specific serum will arise. An alternative approach is to prepare vaccines based on antigenic sites shared among all serotype and subtype viruses.

Type-specific and cross-reactive antigenicity of capsid proteins VP1 and VP2 of echovirus type 7.

After disruption of echovirus type 7 virions with urea and heat, VP1 and VP2 were separated by isoelectric focusing in urea-containing sucrose gradients. Antisera to these two polypeptides were produced in guinea pigs. In complement fixation, antiserum to VP1 reacted with native and heated virions (N and H antigens, respectively) of homologous virus, and also cross-reacted with heated virions of some other enteroviruses used. Antiserum to VP2 was reactive only with heated virions of homologous and heterologous viruses. Interestingly, the anti-VP2 serum reacted neither with native nor even with heated procapsids (naturally-occurring empty capsids). Antiserum to VP1, but not VP2, showed neutralizing and hemagglutination-inhibiting activities. These results suggest that 1) both VP1 and VP2 possess cross-reactive antigenic determinants which are exposed on the surface of heated virions, and 2) type-specific determinants of VP1 are located on the surface of native virions.

A study of the cross-reacting antigens on the intact foot-and-mouth disease virus and its 12S Subunits with antisera against the structural proteins.

Cross-reactions between two strains of foot-and-mouth disease virus (FMDV) belonging to different serotypes (A and O) were studied with intact virus and virus subunits and antisera produced against the isolated structural proteins. Anti-VP1 type O serum showed cross-reactive neutralizing activity, in contrast to the sera raised against intact virus type O, whereas anti-VP1 type A serum only neutralized homologous virus. Anti-VP2, VP3 and VP4 did not show neutralizing activity. In the enzyme-linked immunosorbent assay and radio-immunoassay anti-VP1, -VP2, and -VP3 sera reacted with the 12S virus subunits of both serotypes. No activity was obtained against VP4. Competition experiments with virus subunits of virus type A and O show that anti-VP1 serum is the most type-specific. Anti-VP2 serum is completely cross-reactive, while anti-VP3 serum reacted in an intermediate way. The identical reactions obtained with anti-VP2 sera and the homologous and heterologous virus subunits suggest that the exposed VP2 antigens are identical.

Intracellular localization of viral polypeptides during simian virus 40 infection

African green monkey kidney cells infected by simian virus 40 were analyzed by immunofluorescence techniques for the nature and the time course of the appearance of viral polypeptides during infection. Reagents used in the study were anti-Vpl sera and affinity-purified anti-Vpl immunoglobulin G, anti-Vp3 sera, antivirus (anti-V) sera, and anti-tumor antigen sera. The results are summarized as follows. (i) Three types of staining, nuclear, perinuclear, and perinuclear accompanied by cytoplasmic staining, were observed in infected cells in reaction with anti-vpl antibody. In addition, a highly structured staining was observed at the periphery of nuclei of infected cells late in infection. (ii) In reaction with anti-Vp3 serum, the staining was confined within nuclei of cells throughout infection. (iii) Vp1 and Vp3 antigens seem to occupy different spacial regions of the nuclear area in cells. (iv) Vp1 and Vp3 antigens were expressed simultaneously during infection. (v) Centriolar staining observed early in infection paralleled the appearance of tumor (T-) antigen until 24 h after infection, after which time the frequency of positive centriolar staining decreased as infection progressed. (vi) T-antigen was first expressed at about 8 h after infection, and Vp1 and Vp3 antigens were first expressed at about 20 h after infection.

Vp1 affects intracellular localization of Vp3 polypeptide during simian virus 40 infection.

In order to understand the functions of simian virus 40 genes, permissive cells (TC7) were infected with mutants temperature sensitive in the complementation groups A, B, C, BC, and D at permissive and nonpermissive temperatures. Cells were examined for the localization of viral polypeptide antigens by immunofluorescent staining with monospecific antibodies. The results are as follows: (i) The appearance of Vp1 antigen in cells infected by tsB, C, or BC mutants was not affected appreciably by the mutations. (ii) The appearance of Vp3 antigen was affected by the mutations in B, C, or BC. Vp3 antigen is confined to the nuclei in cells infected by wild-type virus. With mutant virus infection, Vp3 antigen is found in the cytoplasm, perinuclear region, and nucleoli. (iii) The tsD mutants and the tsA mutants did not express either Vp1 or Vp3 antigens at the nonpermissive temperature. (iv) Nucleoli seem to play an essential role in the biosynthesis and assembly of viral polypeptides. Thus, mutations in any one of complementation groups B, C, or BC, which are within the structural gene for Vp1, cause an alteration of intracellular distribution of another late gene product, Vp3. These results suggest that the amino acid sequences of Vp1 polypeptide play a role(s) in the transport of viral antigens across internal membranes or in virus assembly processes or in both.