Yasuo Ichihashi

Subclass of IgG antibody to GM1 epitope-bearing lipopolysaccharide of Campylobacter jejuni in patients with Guillain-Barré syndrome

Sera of patients who develop Guillain-Barré syndrome (GBS) subsequent to Campylobacter jejuni enteritis frequently have IgG anti-GM1 antibody. Lipopolysaccharide (LPS) of C. jejuni isolated from a GBS patient has a GM1 ganglioside-like structure. IgG subclass distribution of the anti-GM1 antibody in GBS patients is mainly restricted to IgG1 and IgG3. Since IgG antibodies to bacterial polysaccharide generally are restricted to IgG2 subclass, some investigators have assumed that either the general rules for immune response to LPS are broken in the patients or an alternative antigen has yet to be identified. To clarify whether the LPS participates in the production of the anti-GM1 antibody, we investigated the subclass of IgG antibody to the LPS that bears GM1-like structure. The subclasses of IgG antibody to the LPS were restricted predominantly to IgG1 and IgG3. The GM1 epitope-bearing LPS may function in the production of the anti-GM1 antibody in patients with GBS subsequent to C. jejuni infection.

Hemadsorption and fusion inhibition activities of hemagglutinin analyzed by vaccinia virus mutants

Vaccinia virus IHD-J strain induces hemagglutinin (HA) on the surface membrane of infected cells and does not elicit cell-cell fusion (F-). We isolated 21 independent hemadsorption-negative (HAD-) mutant viruses from IHD-J and five HAD+ revertants from one of these mutants. Of the 21 mutants, 19 that synthesized either no or little HA at the cell surface caused cell-cell fusion (F+), whereas none of the five revertants that synthesized HA at the cell surface induced cell-cell fusion. Furthermore, anti-HA monoclonal antibody B2D10 induced extensive polykaryocytosis of IHD-J-infected cells and suppressed the ability of the IHD-J-infected cell extract to inhibit the polykaryocytosis induced by IHD-W. The other 2 of the 21 HAD- mutants, B1 and A2, which induced HAs at the cell surface, showed F- and F+ phenotype, respectively. The HA molecule of mutant B1 had a single amino acid substitution of Lys for Glu-121 in its extracellular domain, whereas that of mutant A2 had a single substitution mutation of Tyr for Cys-103. We conclude that the vaccinia HA is a fusion inhibition protein, that the active sites for the two activities reside separately in its extracellular domain, and that cysteine-103 is important in forming the proper tertiary structure of the protein to exert both activities.

The activation of vaccinia virus infectivity by the transfer of phosphatidylserine from the plasma membrane

Purified vaccinia virus usually contains a large proportion of noninfectious virus which can be converted to infectious virus by incubation with purified plasma membrane. This activating reaction which is mediated by a heat stable component of the membrane has been studied. A suspension of liposomes containing the lipids extracted from plasma membrane of either KB cells or mouse RBCs activated the noninfectious virus in the same manner as heated plasma membrane. The phospholipid fraction of the KB cell lipids had the activating ability, but neither neutral lipid nor glycolipid fraction activated the virus. Liposomes containing phosphatidylserine activated the virus, whereas other tested phospholipids, including phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and sphingomyelin had no effect on virus infectivity. Lysolecithin reduced the infectivity. Treatment with isolated plasma membrane or liposomes increased hydrophobicity of the virus slightly, but did not change its density. Analysis of activated and then purified virus showed that all phospholipid species in the coincubated plasma membranes and liposome samples were transferred to the virus. The transfer was not a phospholipid exchange reaction but a one-way net transfer, and took place rapidly at 37 degrees to reach saturation within 1 hr of coincubation. Neither activation of virus nor transfer of phospholipid occurred when the mixture was incubated at a temperature below 8 degrees. The virus has great ability to extract phospholipids from coincubated lipid bilayer membranes, and association with phosphatidylserine gives the virus high infectivity.

Vaccinia-specific hemagglutinin.

Abstract Vaccinia-specific hemagglutinin (VHA) was reconstituted with the protein and phospholipid fractions prepared from vaccinia virus-infected cell membranes by treatment with sodium deoxycholate. The phospholipids of the IHD-J-strain virus (VHA+)-infected cells were replaceable in the constitution by pure phospholipids. In contrast, the protein fraction of VHA−-strain virus-infected cells or of uninfected HeLa cells showed no VHA activity after mixed dialysis with the phospholipid fractions. In addition to the VHA activity, heat stability and blocking capacity against specific anti-VHA serum were also regenerated when the VHA+ protein was combined with phospholipids. The protein fraction of the VHA possessed all the specificities of vaccinia hemagglutinin. Three VHA-specific glycoproteins were identified (VHA P1: molecular weight (MW), 1.5 × 105; VHA P2: MW, 3.4 × 104; VHA P3: MW, 1.2 × 104) by sodium lauryl sulfate (SDS)-polyacrylamide-gel electrophoresis of VHA bound to chick erythrocytes. VHA P2 was found to be a subunit of VHA Pl. Host cell membrane protein (MW, 2.4 × 104), seems to be shifted to VHA P2 by VHA-specific glycosylation. The appearance of VHA activity on host cell plasma membranes is thus dependent on the VHA-specific glycosylation and polymerization of host glycoprotein and on the integration of VHA P1 in the membrane phospholipid bilayer.

The function of the vaccinia hemagglutinin in the proteolytic activation of infectivity

The vaccinia virus hemagglutinin (HA) has specific affinity for the structural protein, VP37K. The nature of this affinity and its relationship to the function of the HA were analyzed using HA mutants. The VP37K reactive site of the HA molecule is located in its transmembrane region, and the vaccinia virus HA associates with the viral particle via the VP37K-HA affinity. The viruses possessing an HA with fusion inhibitor activity were largely of the low infectivity form, whereas the viruses that associated mutant HAs defective in the activity were of the high infectivity form. D1 mutant virus does not produce HA. When it was incubated with the HA of the IHD-J strain, the HA associated with the virus particle. The HA-loaded D1 mutant virus acquired a high affinity not only for chick erythrocytes but also for KB and Vero cells. At the same time, the infectivity for Vero cells was decreased. The original high infectivity was recovered by treatment with trypsin. The virion-associated vaccinia HA has two functions; the HA protects the infectivity of the virus by the fusion inhibitor activity and exhibits affinity against host cells. Vaccinia virus first adsorbs to the cell via HA, and then proteolysis of the HA activates the second adsorption site which seems to be the fusogenic site of the virus. Proteolytic activation represents removal of the fusion inhibitor activity of the HA.

Proteolytic activation of vaccinia virus for the penetration phase of infection

Abstract Treatment of vaccinia virus strain IHD-J with trypsin enhanced the titer by specific activation of the infecting efficiency of the virus, not by dispersion of aggregated virus. When the penetration step was performed in the presence of an inhibitor of serine protease (PMSF), the titer of untreated virus was reduced, whereas trypsin-treated virus infected effectively even in the presence of the drug. There is a PMSF-sensitive step in the viral uncoating process, the function of which is replaceable by treatment of the virus with trypsin. Using PMSF sensitivity and trypsin-catalyzed activation, vaccinia virus populations were classified into three fractions differing in infectivity: a fraction that is infectious in the presence of PMSF (activated form), a fraction that is not infectious in the presence of PMSF but is infectious under usual tissue culture conditions (infectious form), and a fraction that is not infectious under usual conditions and requires treatment with trypsin to become activated (protected form). The ratios in a purified virus sample were 1:3:16, respectively. The ratios of the three forms assayed at various times after infection showed that the activated form and/or infectious form was dominant until 20 hr and the protected form was the large majority after 24 hr of infection. The progeny virus in the medium was of the activated form.

The effect of proteolytic enzymes on the infectivity of vaccinia virus

Abstract Treatment of vaccinia virus strain IHD-J with proteolytic enzymes such as trypsin, chymotrypsin, thermolysin, pronase E, and papain did, while protease V8 did not, increase its infectivity. Virus treated with the enzymes at their optimal dose for activation were resistant to DNase; the membrane and core structures remained intact. Proteins associated with the viral membrane (VP110K, VP88K, VP54K, VP34K, VP32K, and VP14K) were digested at different rates depending on the enzyme used; but proteins in the core region retained their molecular weight. The proteolytic activation of viral infectivity is related to a change in the virus surface proteins. To examine the viral proteins of enzyme-treated virus, the viral proteins were blotted onto nitrocellulose sheets after SDS-PAGE, and stained with antisera and FITC-Protein A. The proteins specifically reactive with hyperimmune anti-IHD-J serum were VP43K and VP41K of intact virus, the 41K cleavage product in trypsin-treated virus, and the 43K and 36K of papain-treated virus. Immunestaining profiles of virus treated with suboptimal enzyme doses showed that these proteins were derived from VP88K. The 30K protein detected in all enzyme-activated virus did not react with hyperimmune serum. Peptide analysis indicated that the 30K protein was a cleavage product of VP34K, and that confirmed that VP88K possessed the same peptides as VP43K. Our findings suggest that the cleavage products derived from VP88K are the activated penetration factors for the first phase of uncoating.

Epitope mosaic on the surface proteins of orthopoxviruses

Epitopes on the surface components of orthopoxviruses were analyzed with monoclonal antibodies (MAbs) against monkeypox and vaccinia viruses by enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), radioimmunoprecipitation (RIP), and competitive binding inhibition assay (CBIA). When compared by ELISA, three vaccinia virus strains exhibited a similar reactivity to 99 tested MAbs despite their remote passage history. All five isolates of monkeypox virus closely resembled one another, irrespective of the host species (human, monkey, squirrel) from which they were isolated. Taterapox virus reacted similar to vaccinia virus against 97 of the 99 tested MAbs, and reacted with 2 MAbs which were cross-reactive with monkeypox and mousepox. Mousepox and cowpox viruses reacted with these MAbs in a species-specific manner: MAbs reactive to cowpox virus distinctly differ from those reactive to mousepox virus. Of the 99 tested MAbs, 32 reacted with all the 11 tested orthopoxviruses, indicating that the corresponding epitopes existed in all the viruses. Fifty-four MAbs reacted with two or more virus species and were classified as partially common MAbs. Eight MAbs were apparently type-specific for monkeypox, and five were specific for vaccinia and taterapox viruses. No strain-specific epitope was detected. Sera of monkeypox-infected patients, when analyzed by CBIA, interfered with the binding of monkeypox-specific MAb H12C1 but not of vaccinia-specific MAb G6C6. Sera of monkeypox-infected patients who had been vaccinated competed against both MAbs, demonstrating the original antigenic sin phenomenon. The two MAbs could distinguish between the sera of monkeypox patients and those of vaccinated persons. However, the serum of a smallpox patient was competitive against these apparently vaccinia- or monkeypox-specific MAbs. Three of the eight monkeypox-specific epitopes were recognized by the above CBIA test, which suggests that they also exist in smallpox virus. The mosaic-like combination of common epitopes and the small number of type-specific epitopes manifested the antigenic characteristics of orthopox viruses. The species boundary was obscured due to the partially common epitopes, but the total composition of epitopes was stable enough to maintain the antigenic species-specificity. The mutual relationship of the orthopoxviruses was visualized in a three-dimensional network.

Modification of vaccinia virus penetration proteins analyzed by monoclonal antibodies

Modifications induced in structural vaccinia virus proteins that elicit the high infectious state by virus activating treatments involving trypsin and phosphatidylserine were analyzed using antivaccinia monoclonal antibodies (MABs). MABs reactive against each of the five outer layer proteins (VP54K, 34K, 32K, 29K, and 17K-25K) neutralized infectivity. VP54K possesses at least two neutralizing epitopes. Treatment with trypsin or with isolated plasma membrane cleaved VP54K into TVP41K carrying epitope A and removed a fragment containing epitope B from the virus. MABs against either of the epitopes could neutralize the virus. The exposure of epitope A concomitantly activated virus infectivity, and it was an essential step of penetration. MABs against VP17K-25K reacted more efficiently with trypsin-treated virus than with untreated virus, but the size of VP17K-25K was not affected by trypsin; this finding indicated that trypsin treatment rendered the VP17K-25K epitopes more accessible to antibody and hence to neutralization. MABs against VP32K and VP29K neutralized infectivity to the same extent irrespective of the state of activation. Virus treated with phosphatidylserine (PS) was neutralized more efficiently by MAB against VP34K than untreated virus, but the amount of antibody that reacted with the virus was the same before and after treatment with PS. Phosphatidylserine did not modify epitope structure itself, but it activated the function of VP34K. It was concluded that blocking of the functions attributed to any of the five proteins resulted in neutralization of virus infectivity, and treatment with trypsin and phosphatidylserine activates infectivity of vaccinia virus by modifying three of them (VP54K, VP34K, VP17K-25K) with characteristic behavior for each protein.

Target Antigen of Vaccinia‐Infected Cells Recognized by Virus‐Specific Cytotoxic T Lymphocytes

A vaccinia‐specific target antigen for recognition of anti‐vaccinia cytotoxic T lymphocytes (CTL) was found to be formed on the surface of infected cells through two distinct processes. In the first phase, the expression of the target antigen was dependent on the dose of inoculated virus, without specific protein synthesis. The target antigen seems to be produced by virus‐cell fusion. In the second phase, the expression of the target antigen was accompanied by synthesis of an early protein. In spite of the difference in their mode of expression, the first‐phase and the second‐phase target antigens were cross‐reactive in cytotoxicity inhibition assays.