Expression of the structural proteins of dengue 2 virus and yellow fever virus by recombinant vaccinia viruses

Summary Vaccinia virus recombinants were constructed which contained cDNA sequences encoding the structural region of dengue 2 virus (PR159/S1 strain) or yellow fever virus (17D strain). The flavivirus cDNA sequences were expressed under the control of the vaccinia 7.5k early/late promotor. Cultured cells infected with these recombinants expressed immunologically reactive flavivirus structural proteins, precursor prM and E. These proteins appeared to be cleaved and glycosylated properly since they comigrated with the authentic proteins from dengue 2 virus- and yellow fever virus-infected cells. Mice immunized with the dengue/vaccinia recombinant showed a dengue-specific immune response that included low levels of neutralizing antibodies. Immunization of mice with the yellow fever/vaccinia recombinant was less effective at inducing an immune response to yellow fever virus and in only some of the mice were low titers of neutralizing antibodies produced.     

Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins

Summary Recombinant Hantaan virus nucleocapsid protein (rNP) and recombinant envelope (rEnv) proteins were prepared using a baculovirus expression system to examine the role of Hantaan virus structural proteins in protective immunity. Passive transfer of spleen cells from mice immunized with rNP conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD₅₀ (survival rate: 43%) or 4 LD₅₀ (survival rate: 43%). The T cell-enriched fraction protected one mouse from lethal infection but the B cell-enriched fraction had no such effect on fatal HTN infection. The protective effects of the antibody against HTN challenge were examined by passive immunization. The monoclonal antibody ECO 2 directed to NP also conferred partial survival and significant difference in time to death. Although rEnv antigen failed to induce neutralizing antibody, both immune spleen cells and immune serum to rEnv conferred partial protection upon suckling mice. These results indicate that both nucleocapsid and envelope proteins of Hantaan virus were responsible for induction of cell mediated protective immunity. Vero E 6 cells infected with Hantaan virus expressed envelope protein on the surface, as determined by flow cytometry. However, there was only negligible expression of nucleocapsid protein.     

Antivirus antibody-dependent cell-mediated cytotoxicity during murine cytomegalovirus infection.

BALB/c mice infected with murine cytomegalovirus were studied to determine whether antibody-dependent cell-mediated cytotoxicity contributes to the immune control of this infection. Antibody-dependent killer cells from uninfected mice were used as effector cells to assay for antibody in sera of infected mice. Secondary immune sera were found to contain both cytomegalovirus-specific and autoreactive antibodies. After primary infection only cytomegalovirus-specific antibodies were found. These were detected by antibody-dependent cell-mediated cytotoxicity within 8 to 10 days after onset of infection, but usually not until day 21, by a neutralizing antibody assay. Antibody titers were about 10-fold higher by antibody-dependent cell-mediated cytotoxicity than by neutralization. The results indicate that cellular immunity to cytomegalovirus infection includes an antibody-dependent cell-mediated cytotoxicity response which is likely to be highly efficient and may contribute significantly to control of both acute and later stages of infection.     

Inhibition of CD95 (Fas/Apo1)-mediated apoptosis by vaccinia virus WR

Stimulation of the CD95 (Apo-1/Fas) molecule either by the CD95 ligand or by monoclonal antibodies induces programmed cell death by apoptosis in a variety of cell lines and primary cells. In this study we observed that infection of B lymphoblast and T lymphoblast cell lines with vaccinia virus strain WR and recombinant vaccinia WR constructs, but not strain Copenhagen, rendered cells refractory to CD95-mediated apoptosis. In particular, vaccinia virus infection suppressed anti-CD95 antibody-induced membrane disintegration, apoptotic nuclear morphology of cells, and DNA fragmentation. Inhibition of apoptosis was not mediated by CD95 down-regulation or reduced binding of anti-CD95 antibody to infected cells, and occurred at a time point when cellular metabolism was not yet affected by the lytic vaccinia virus infection. Vaccinia virus (WR)-infected cells were resistant to CD95 ligand–CD95-mediated lysis by CD4⁺ and CD8⁺, T lymphocytes. Because cytolysis mediated by CD95 is one of two major mechanisms used by cytotoxic T lymphocytes to kill target cells, inhibition of CD95-mediated apoptosis may constitute a novel immune escape mechanism for this virus. Additionally, this mechanism may contribute to the higher pathogenicity of vaccinia virus strain WR compared with strain Copenhagen.     

Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies

The vaccinia virus entry/fusion complex (EFC) is comprised of at least eight transmembrane proteins that are conserved in all poxviruses. However, neither the physical structure of the EFC nor the immunogenicity of the individual components has been determined. We prepared soluble forms of two EFC components, A28 and H2, by replacing the transmembrane domain with a signal peptide and adding a polyhistidine tail. The proteins were expressed by baculoviruses, secreted from insect cells, purified by affinity chromatography and used to raise antibodies in rabbits. The antibodies recognized the viral proteins but only the antibody to recombinant A28 bound intact virions and neutralized infectivity. Analyses with a set of overlapping peptides revealed a neutralizing epitope between residues 73 and 92 of A28. Passive immunization of mice with IgG purified from the anti-A28 serum provided partial protection against a vaccinia virus intranasal challenge, whereas IgG from the anti-H2 serum did not.     

Infections of Congenitally Athymic (Nude) and Normal Mice with Avirulent and Virulent Strains of Venezuelan Encephalitis Virus

Two strains of Venezuelan encephalitis virus that are avirulent for normal BALB/c mice inoculated subcutaneously were also avirulent for infected congenitally athymic (nude) mice of the same strain. Viremias were of similar magnitudes and durations in normal and nude mice. Brain concentrations were higher in nude mice with the one avirulent strain tested, although the periods of detectable virus in brains were similar. No lesions were found in brains, spleens, or lymph nodes by ordinary histopathological examination. Viral neutralizing antibody titers in plasmas at 1 to 3 weeks after infection were lower and more transient in nude than in normal mice, and implantations of thymic tissues into nude mice partially restored their neutralizing antibody responses. Concentrations of spleen cells producing antibodies that lysed sheep erythrocytes 4 days after inoculation of erythrocytes and avirulent virus into nude mice were above the levels of uninfected nude mice. These concentrations were similar in infected and uninfected normal mice. In contrast, two mouse-virulent strains of Venezuelan encephalitis virus killed nude mice faster than normal mice after subcutaneous inoculation. Yet concentrations and durations of virus in bloods and brains were not consistently different between nude and normal mice. There were perivascular monocytes in brains and slight architectural alterations of spleens and lymph nodes. Concentrations of spleen cells producing antibodies hemolytic for sheep erythrocytes 4 days after inoculation with erythrocytes were depressed in nude and normal mice by infection with virulent strains.     

Kinetics of Epstein-Barr Virus (EBV) Neutralizing and Virus-Specific Antibodies after Primary Infection with EBV

Prospective studies of antibodies to multiple Epstein-Barr virus (EBV) proteins and EBV neutralizing antibodies in the same individuals before, during, and after primary EBV infection have not been reported. We studied antibody responses to EBV in college students who acquired primary EBV infection during prospective surveillance and correlated the kinetics of antibody response with the severity of disease. Neutralizing antibodies and enzyme-linked immunosorbent assay (ELISA) antibodies to gp350, the major target of neutralizing antibody, reached peak levels at medians of 179 and 333 days after the onset of symptoms of infectious mononucleosis, respectively. No clear correlation was found between the severity of the symptoms of infectious mononucleosis and the peak levels of antibody to individual viral proteins or to neutralizing antibody. In summary, we found that titers of neutralizing antibody and antibodies to multiple EBV proteins increase over many months after primary infection with EBV.     

Vaccinia virus immune evasion

Vaccinia virus expresses many virulence factors that are non-essential for virus replication in cell culture but are important in vivo. In this paper three mechanisms are described that are used by vaccinia virus to evade the host immune response to infection. One of these is the release of a soluble protein that binds CC chemokines and that is unrelated to cellular chemokine receptors. The other two mechanisms are displayed by virus particles that are released from infected cells. This form of vaccinia virus is called extracellular enveloped virus (EEV) and is resistant to neutralisation by antibody and to destruction by complement. Resistance to complement is mediated by the acquisition of host complement control proteins, particularly CD55, during virus release from infected cells.     

Protection of mice against vaccinia and herpes simplex virus infection by Propionibacterium acnes

Heat-killed cell suspension of several Propionibacterium acnes strains were prepared and studied for their protective activity in viral infection of mice and immunomodulating properties. Majority of the strains caused marked spleen enlargement in the treated mice. These changes persisted for several weeks. Only some of the tested strains enhanced significantly primary humoral immune response against sheep red blood cells. There was no increase, however, in neutralizing or hemagglutination-inhibition antibody production against vaccinia virus in mice treated with Propionibacteria. No evidence in the increase of spleen lymphocytes migration inhibition of mice infected with vaccinia virus and treated with P. acnes or in hypersensitivity reaction to oxazolone have been found. Significant resistance enhancement of mice pretreated with P. acnes against vaccinia virus or herpes simplex virus type 1 infection was observed. Activity of Propionibacteria depended on the applied strain, dose and scheme of administration.     

Vaccinia virus proteins on the plasma membrane of infected cells. III. Infection of peritoneal macrophages

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some "early" virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted; with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no "late" virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody-125I-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48-50, 45-46, 36-37, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells.     

Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody.

Studies were performed to determine whether active immunity against murine rotavirus (EDIM) infection of mice correlated with titers of neutralizing antibody to the challenge virus. Neonatal mice administered either murine or heterologous rotaviruses all developed diarrhea and high titers of serum rotavirus IgG. However, only mice given EDIM, the murine EB, or simian SA11-FEM strains were protected against EDIM infection when challenged 60 days later. Other serotype 3 strains (RRV, SA11-SEM), as well as strains belonging to serotypes 5 and 6 (OSU, NCDV, WC3), were not protective. Serum neutralizing antibody titers to EDIM were almost undetectable after rotavirus infection with any strain and could not, therefore, be correlated with protection. Likewise, intestinal neutralizing antibody titers were extremely low 21 days after EDIM infection, and by 60 days after inoculation, EDIM-infected mice had no greater intestinal neutralizing antibody titers than uninoculated controls. Mice inoculated with SA11-FEM as neonates had much higher serum rotavirus IgG responses than mice inoculated as adults, and only those infected with this virus as neonates were protected. Thus, although immunity to EDIM did not correlate with the presence of neutralizing antibody to EDIM, it did correlate with the overall magnitude of the immune response after inoculation with SA11-FEM.     

表达HPV18E7E6的重组痘苗病毒构建及免疫效果的初步研究

目的 构建表达HPV18E7E6融合蛋白的重组痘苗病毒,并对E7E6蛋白的免疫原性进行研究.方法 将去除了转化活性的HPV18E6、E7基因融合,插入痘苗病毒重组质粒,通过同源重组构建表达HPV18E7E6的重组痘苗病毒,观察其免疫效果.结果 构建了表达E7E6融合蛋白的重组痘苗病毒,PCR鉴定及测序表明融合基因序列与设计相符,正确插入到痘苗病毒TK区域;Western-Blot检测表明该重组病毒能表达HPV18E7E6融合蛋白.免疫后的小鼠可产生E6、E7特异性抗体,但ELISPOT没检测到E7肽库刺激小鼠脾细胞产生分泌IFN-丫的阳性反应.结论 构建了一株表达HPV18E7E6融合蛋白的重组痘苗病毒,可以有效诱发小鼠产生针对E6、E7的体液免疫,但不能诱发产生相应的细胞免疫,为进一步研究不同动物模型中HPV18E6E7的细胞免疫特点提供了实验基础.     

Virus-specific major MHC class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection

A transgenic mouse expressing MHC class II-restricted TCR with specificity for a lymphocytic choriomeningitis virus (LCMV) glycoprotein-derived T helper cell epitope was developed to study the role of LCMV-specific CD4+ T cells in virus infection in vivo. The majority of CD4+ T cells in TCR transgenic mice expressed the transgenic receptor, and LCMV glycoprotein-specific TCR transgenic CD4+ T cells efficiently mediated help for the production of LCMV glycoprotein-specific isotype-switched antibodies. In contrast, LCMV glycoprotein-specific TCR transgenic mice exhibited a drastically reduced ability to provide help for the generation of antibody responses specific for the virus-internal nucleoprotein, indicating that intramolecular/intrastructural help is limited to antigens that are accessible to B cells on the viral surface. Antiviral cellular immunity was studied with noncytopathic LCMV and recombinant cytopathic vaccinia virus expressing the LCMV glycoprotein. TCR transgenic mice failed to efficiently control LCMV infection, demonstrating that functional LCMV-specific CD4+ T cells – even if activated and present at extremely high frequencies – cannot directly mediate protective immunity against LCMV. Despite the fact that LCMV-primed CD4+ T cells from TCR transgenic mice as well as from control mice showed low MHC class II-restricted cytotoxic activity in vivo, this did not correlate with protection against LCMV replication in vivo. In contrast, CD4+ T cells from TCR-transgenic mice mediated efficient protection against infection with recombinant vaccinia virus. These results further support the need for different immune effector functions for protective immunity against different viral infections.     

The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses

In vitro, T cell-dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin-, the other Fas-dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the noncytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin-dependent mechanism without measurable involvement of the Fas-dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin-dependent cytotoxicity mediated by T cells is crucial for protection against noncytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell-dependent soluble mediators such as cytokines (IFN-γ against vaccinia virus) and neutralizing antibodies (against VSV and SFV).     

Demonstration of Cytotoxic Antibodies in Rabbits Bearing Tumors Induced by Shope Fibroma Virus

The (51)Cr-release test was used to detect cytotoxic antibodies in adult rabbits bearing tumors induced by Shope fibroma virus. The following are the recommended experimental conditions: the infection of RK-13 cells with a multiplicity of 1 to 2 infectious units per cell for 48 hr, (51)Cr labeling of infected cells during the last 12 hr of incubation, sensitization of suspended labeled infected cells for 1 hr with immune serum, and quantitation of cell damage by the amount of (51)Cr released after 6 hr of incubation in the presence of complement. The immune sera reacted only with fibroma virus-infected cells but not with cells infected with vaccinia virus or herpesvirus type 1. Similarly, sera prepared against vaccinia virus and herpesvirus type 1 were not cytotoxic to fibroma virus-infected cells, although they were cytotoxic to cells infected with homologous viruses. The total antibody activity in sera of rabbits infected with Shope fibroma virus was detected first on day 7, gradually rose to its peak by day 23, and persisted at that level for at least 50 days. The 19S antibody was detected on day 7, reached peak titers by day 13, and disappeared by day 17. The 7S antibody was barely detectable on day 7, reached maximum titers on day 13, and remained high for at least 50 days. The tumors appeared on the 3rd day after virus inoculation, reached maximum size on day 13, and regressed completely by day 23.     

Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus

BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody-LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti-LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus.     

Neutralizing antibody formation in swine infected with seven strains of porcine reproductive and respiratory syndrome virus as measured by indirect ELISA with peptides containing the GP5 neutralization epitope

An indirect ELISA with peptides containing the GP5 neutralization epitope was used to measure the time courses of formation of neutralizing antibodies in sera of groups of 10 pigs infected with seven different strains of porcine reproductive and respiratory syndrome virus (PRRSV) generated in an earlier study (Johnson et al., Vet.Immunol. Immunopath. 102:233-247. 2004). The neutralizing antibody responses varied greatly between individual pigs infected with each PRRSV strain. Some pigs generated high titers of neutralizing antibodies between 7 and 28 days post infection (p.i), whereas other pigs had not generated a significant response by 42 days p.i. The heterogeneity in antibody formation in individual pigs also pertained to anti-N-protein antibody formation as measured by both HerdCheck and peptide ELISAs and there was no correlation between anti-N-protein and neutralizing antibody formation. Also, viremia was disconnected from neutralizing antibody formation. Viremia peaked 7-15 days p.i. and then precipitously declined to undetectable levels in most infected pigs by 21-28 day p.i. whether or not they had generated neutralizing antibodies. Furthermore, contrary to an earlier conclusion from this study the formation of neither neutralizing nor anti-N-protein antibodies was related to viral load as measured by infectious virus levels in serum.     

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by an intrahepatic inoculation with an expression plasmid

We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically or intramuscularly inoculated with an expression plasmid DNA encoding HCV structural proteins under the control of the elongation factor 1-alpha promoter. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. CTL responses were examined using target cells either pulsed with a specific peptide or infected with a recombinant vaccinia virus expressing HCV structural protein. Both intrahepatically and intramuscularly DNA-inoculated mice developed CD8(+), MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells. These studies demonstrated the usefulness of a murine model of HCV infection induced by direct intrahepatic DNA inoculation for understanding the immunopathogenic mechanisms in HCV infection.     

Potentiation of coxsackievirus B3 infection in adult mice pretreated with a gold salt.

In mice treated with sodium aurothiomalate (myocrisin), prior to infection with Coxsackievirus B3, 90% of the animals died by the 11th day postinfection (p.i.). A mortality of 10% was noted in mice receiving myocrisin only, and no deaths occurred in animals infected with virus alone. The highest amount of virus was recovered from the pancreas of myocrisin-treated mice on day 3 p.i. This was over 500-fold higher than the virus titer found in the pancreas of mice infected with virus only. Generally the titer of virus present in different organs was higher at every point in drug-treated animals as compared to intact mice infected with the virus. A high and persistent viremia was present in myocrisin-treated mice; in contrast a low viremia followed by virus clearance from the blood was observed in intact mice infected with the virus. The antibody response was studied in intact and myocrisin-treated mice infected with the virus. In both groups, no neutralizing antibodies were detected on days 1, 2, and 3 p.i. On day 7 after infection, the titers of antibodies were 1:16 and 1:12 in intact and myocrisin-treated mice, respectively. Administration of hyperimmune anti-Coxsackievirus B3 serum 6 hours after infection protrected in myocrisin-treated group of mice against lethal disease. The results of these studies suggest that (1) antibodies alone may not be sufficient to limit the spread and persistence of virus in natural infections and (2) in the absence of any apparent histopathological differences the increased multiplication of Coxsackievirus B3 could be the cause of death in myocrisin-treated mice.     

Response of dairy calves to vaccinia viruses that express foreign genes.

Repeated intradermal inoculations of calves with wild-type vaccinia virus and recombinant vaccinia viruses expressing human hepatitis B virus surface antigen and herpes simplex virus, type 1, glycoprotein D produced characteristic pox lesions at each site of injection. In some instances, calves were inoculated as many as five times at intervals from 4 to 7 weeks. The lesions invariably were more severe after the second inoculation. Subsequent inoculations produced a less severe area of redness, swelling, necrosis, and scab formation. No other signs of illness, such as an elevation in temperature, were noted in the calves. Vaccinia virus was isolated in low titers from scabs taken at various times after inoculation. No lesions were formed at the sites injected with tissue culture fluid and cellular debris at the same time that virus inoculations were made. Calf contact controls remained normal through the 8-week exposure in isolation units with calves inoculated twice with vaccinia virus. No neutralizing antibody to vaccinia virus was detected in the contact controls. In contrast, the virus-inoculated calves developed neutralizing antibody to vaccinia virus and to herpes simplex virus glycoprotein D in serum. In all cattle, a second inoculation significantly enhanced the neutralizing antibody response within 1 week, suggesting that an anamnestic response had occurred. No antibody to hepatitis B virus surface antigen was elicited in calves after repeated inoculations with vaccinia recombinants that express hepatitis B virus surface antigen and are known to elicit in rabbits antibodies reactive with hepatitis B virus surface antigen.