- Volume 73, Issue 9, 1992
Volume 73, Issue 9, 1992
- Animal
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Measles virus from a long-term persistently infected human T lymphoblastoid cell line, in contrast to the cytocidal parental virus, establishes an immediate persistence in the original cell line
More LessTo investigate the mechanisms of measles virus (MV) establishment and maintenance of persistence in lymphoid cells, we have established a long-term persistent infection with MV, Edmonston strain, in the human T lymphoblastoid cell line MOLT4, which has been in continuous culture for over 8 years. In this culture, designated MOMP1, more than 98% of cells display viral antigens. The MOMP1 culture is immune to superinfection with MV and is not cured by anti-MV antibodies. No evidence of defective interfering particles was obtained. The persistently infected culture releases an infectious virus showing a miniplaque and thermoresistant modified phenotype that, unlike the parental virus Edmonston strain which produces a lytic infection with extensive cell fusion, establishes an immediate persistence in MOLT4 cells with neither significant loss of cell viability nor cell fusion. This suggests that the modification in the virus suffices to maintain the state of persistence without requiring a coevolution of the host cell during the infection, as has been reported in other persistent virus infections.
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Measles virus gene expression in lytic and persistent infections of a human lymphoblastoid cell line
More LessMOMP1 is a measles virus (MV) long-term steady-state persistently infected culture of the human T lymphoblastoid cell line MOLT4. The analysis of MV gene expression revealed that in MOMP1 cells, the major MV proteins, haemagglutinin (H), phosphoprotein (P), nucleoprotein, fusion (F) and matrix (M), are present and the fusion precursor (F0) is cleaved into F2 and F1 peptides. H and F2 proteins are glycosylated in both lytic and persistent MOLT4 infections. All major proteins are underexpressed in the persistently infected cultures in comparison to the lytically infected cells. However a relatively greater reduction was observed for H, M and P proteins. Pulse-chase labelling experiments indicated that this underexpression of H, M and P proteins was not due to selective degradation of these proteins in the persistent infection (p.i.). The relative amounts of the major monocistronic and dicistronic mRNAs for MV proteins, with the exception of P mRNA, was not altered in the p.i. with respect to lytically infected MOLT4 cells, suggesting that the defective expression of H and M proteins was not due to a restriction in the transcription of their mRNAs. In contrast, the mRNA for P protein, the most abundant MV mRNA in these lytically infected T lymphoid cells, is markedly underexpressed in the homologous p.i. Thus the underexpression of P protein in p.i. could be due to a decreased availability of P mRNA. This unbalanced underexpression of MV proteins may impair the cell fusion and c.p.e. of MV and facilitate viral persistence in human lymphoid cells.
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Heterogeneity of linear B cell epitopes of the measles virus fusion protein reacting with late convalescent sera
More LessB cell epitopes of the measles virus fusion protein were mapped, by reacting sera from late convalescent donors with synthetic overlapping pentadecapeptides, segments covering the whole F protein sequence. Unselected individual sera recognized 7 to 20% of the total sequence. Cumulation of the binding patterns of 30 sera identified eight to 10 clusters of antibody-binding peptides spread over most of the sequence. The B cell epitopes included regions of transition between the more hydropathic (including the N-terminal end of the F1 and F2 protein) and hydrophilic sequences. When the regions of antibody binding were compared with the predicted secondary structure of the F protein, no detectable pattern became apparent. Exposed sequences as well as sequences hidden in the viral membrane or in the protein core of both the F1 and F2 polypeptides were recognized by the antibodies. The heterogeneity of the binding patterns was not merely dependent on the anti-measles virus titre. The importance of antibodies recognizing linear epitopes of the measles virus fusion protein for the immune protection is presently not known.
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Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies
G. Taylor, E. J. Stott, J. Furze, J. Ford and P. SoppThe regions of the fusion protein of respiratory syncytial virus (RSV) that react with neutralizing, fusion-inhibiting and highly protective bovine and murine monoclonal antibodies (MAbs) were mapped by two methods: (i) competitive binding assays and (ii) production and analysis of antibody-escape mutants. Competitive binding assays with 16 murine and 10 bovine MAbs identified 11 antigenic sites on the fusion (F) protein, many of which overlapped extensively, and indicated that cattle, a natural host for RSV, and mice recognize similar epitopes. Neutralizing MAbs identified four sites, two of which were also fusion-inhibiting and highly protective in mice. The pattern of reactivity of antibody-escape mutants with the MAbs confirmed the mapping of the protective epitopes deduced from competitive binding assays. A comparison of the biological properties of MAbs to the F protein indicated that protection against RSV infection correlated with fusion inhibition rather than neutralization titre or complement-dependent lysis of virus-infected cells.
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Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus
Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.
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Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies
More LessBacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits. Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation. Two of these antisera were specific for either the amino- or carboxy-terminal regions of the L protein. The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein. Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase. Antigenically authentic L protein was expressed. Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity. Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein. The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay. These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability. Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase.
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Organization of Germiston bunyavirus M open reading frame and physicochemical properties of the envelope glycoproteins
More LessWe describe the construction of plasmids which express fusion proteins representing various regions of Germiston virus M polyprotein. The fusion proteins were purified and inoculated into rabbits to produce antisera. The N- and C-terminal regions of the polyprotein induced specific antibodies which reacted with glycoproteins G2 and G1, respectively, and the intermediate region induced antibodies against the NSM polypeptide. This enabled us to determine the gene order: G2-NSM-G1. Glycoproteins G1 and G2 form the spikes on the surface of the virion. We attempted to determine the structural organization of the glycoproteins by using a membrane-permeable cross-linking reagent, dimethyl suberimidate, but were unable to demonstrate that G1 and/or G2 form oligomeric structures. We analysed the glycoproteins further and showed that, like peripheral membrane proteins, the G2 and NSM proteins are almost completely extracted into the aqueous phase of detergent Triton X114-treated cellular extracts, whereas glycoprotein G1 is distributed in almost equal proportions between the aqueous and the detergent fractions. This indicates that G1 is a membrane-associated protein, but its presence in the aqueous phase suggests that it is less hydrophobic than a typical membrane protein. We have also characterized the intracellular transport of the envelope glycoproteins from the endoplasmic reticulum to the Golgi complex. Pulse-chase labelling followed by immunoprecipitation and treatment with endoglycosidase H (endo H) showed that both G1 and G2 are transported from the endoplasmic reticulum to the Golgi complex. Conversion to the endo H-resistant form is a rather slow process which takes more than 2 h. The mature G1 and G2 proteins present in the virion particle contain almost completely endo-H-resistant glycans.
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Non-random reassortment between the tripartite RNA genomes of La Crosse and snowshoe hare viruses
More LessThe process of reassortment between the tripartite RNA genomes (segments designated L, M and S) of snowshoe hare and La Crosse bunyaviruses (Bunyaviridae) has been investigated by polymerase chain reaction analysis of > 250 progeny recovered at 72 h post-infection from dual wild-type virus infections involving high multiplicities (approximately 5) of each virus in a BHK-21 cell line. Statistical analysis of the data indicated that RNA segment reassortment was not random, and for these two viruses the data appeared to fit the hypothesis that there was a preference for homologous L-M and M-S associations among the progeny formed.
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Immunogenicity and vaccine efficacy of synthetic peptides containing Semliki Forest virus B and T cell epitopes
More LessA synthetic peptide that contains a Semliki Forest virus (SFV) B cell epitope, located at amino acid positions 240 to 255 of the E2 protein, and an SFV T helper (Th) cell epitope, located at positions 137 to 151 of the E2 protein, evoked high titres of SFV-reactive antibodies in H-2d mice. Although the peptide-induced antibodies did not neutralize SFV in vitro, 70 to 100% of the peptide-immunized mice were protected against SFV, even when viral challenge was presented 4 months after immunization. The protection could be transferred by anti-peptide serum, indicating that antibodies were responsible for the protection. When the Th cell epitope of this protective peptide was replaced by an influenza virus Th cell epitope or by another SFV Th cell epitope, the resulting peptides induced lower non-neutralizing SFV-reactive antibody titres and protected a correspondingly lower percentage of mice (50% and 30%, respectively). A peptide with the same Th cell epitope as the best protective peptide but with a less effective SFV B cell epitope protected only 33% of the mice. These results indicate that protection against SFV by a synthetic peptide is primarily dependent on its ability to induce adequate amounts of antibodies with relevant specificity and sufficient affinity; the ability to induce a relevant (SFV-specific) T memory response played only a minor role in protection.
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Potential significance of the cellular immune response against the macaque strain of simian immunodeficiency virus (SIVMAC) in immunized and infected rhesus macaques
The cellular immune response of seven rhesus macaques immunized with Tween-ether-treated macaque strain of simian immunodeficiency virus (SIVMAC) and three non-vaccinated control animals was investigated. Immunization elicited antigen-specific proliferating CD4+ cells in five of seven monkeys. Proliferating T cells were found in all animals protected from a first virus challenge. Cytotoxic T lymphocytes (CTLs) were not induced by the immunization. After the second challenge, the four formerly protected animals became infected, despite a strong proliferative CD4+ cell activity in three of them. All animals lost their proliferative activity 2 weeks after infection. After the first challenge four of the six infected animals exhibited a CTL response and after the second challenge, one of four newly infected macaques acquired a CTL response. The five animals with a CTL activity against SIVMAC proteins were protected from severe thrombo-cytopenia, which appeared in the five CTL-negative animals after infection. Our data show the induction of proliferative T cells by immunization with soluble SIVMAC antigen. This T cell reactivity was found in all animals protected from the first virus challenge, but did not confer protection from the second challenge. Interestingly, the proliferative T cell reactivity disappeared 2 weeks after virus infection. Furthermore a CTL response against viral proteins seems to protect infected animals from severe thrombocytopenia which is an early sign of AIDS in monkeys.
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Human T cell leukaemia virus type 1 p21X mRNA: constitutive expression in peripheral blood mononuclear cells of patients with adult T cell leukaemia
Although the p21X protein of human T cell leukaemia virus type 1 (HTLV-1) is generally thought to be expressed from a doubly spliced mRNA transcript (tax/rex mRNA) that encodes the p40tax, p27rex and p21X proteins, we have shown previously that a novel, alternatively spliced mRNA transcript (p21X mRNA) is responsible for p21X production in HTLV-1-infected cell lines. In the present study, we analysed expression of p21X mRNA and tax/rex mRNA in uncultured and cultured peripheral blood mononuclear cells (PBMCs) from eight patients with adult T cell leukaemia by using a quantitative polymerase chain reaction coupled to reverse transcription. The results demonstrated that the expression of p21X mRNA occurs constitutively in all uncultured and cultured PBMCs, whereas the expression of tax/rex mRNA is inducible in the cultured PBMCs, as described previously. In uncultured and cultured PBMCs from the one specimen in which p21X mRNA was highly expressed, the p21X protein was detectable by Western blotting. On the other hand, p27rex protein was detectable only after cultivation. These findings indicate that p21X mRNA is constitutively expressed in vivo and is responsible for production of p21X protein.
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The effects of poly(I). poly(C12U) and interferon on the multiplication of a mammalian type C retrovirus in human cells
More LessPoly(I).poly(C12U) or interferon treatment inhibited multiplication of the xenotropic baboon type C endogenous retrovirus M7 in chronically infected human AV3-M7 cells, as determined by a reverse transcriptase (RT) assay and electron microscopy. Furthermore, this polynucleotide induced 2′5′ oligoadenylate (2′5′A) synthetase activity. In contrast to interferon (IFN), poly(I).poly(C12U) did not give rise to the appearance of a trapping phenomenon observable by electron microscopy. When AV3-M7 cells were treated simultaneously with poly(I).poly(C12U) and anti-IFN-β/α antibodies, the induction of 2′5′A synthetase was abolished without any alteration of the inhibitory effect of RT activity. Taken together, these results suggest that different mechanisms are used by poly(I).poly(C12U) and IFN in blocking type C retrovirus multiplication.
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Dissociable antiviral activities directed against cardioviruses are expressed in L cells treated with interferon
More LessInterferon (IFN) restricts a wide variety of viruses. To do so it elicits many antiviral pathways. For example, subclones of the same cell line with a reduced antiviral spectrum are thought to lack one or more antiviral pathways. Our line of L cells exhibits two distinct antiviral activities. The first delays the yield of both wild-type mengovirus (is +) and an IFN-sensitive mutant (is-1). The second specifically inhibits is-1 virus yields 100-fold. From these cells, a subclone was isolated which had lost the second antiviral activity (i.e. in these cells is-1 virus acts like is + virus). To see whether other cardioviruses are sensitive to these activities, two additional strains [m-mengovirus and encephalomyocarditis-R (EMC-R) virus] were tested in our subclones. Like is + virus, m-mengovirus yields were delayed by IFN in both subclones; EMC-R virus behaved like is-1 virus in both cell lines. When actinomycin D was added at the time of infection, is-1 virus was phenotypically reversed to is + virus, but EMC-R virus was still inhibited. The 2-5A synthetase/RNase L pathway is expressed in both clones. Therefore, at least three antiviral activities against cardioviruses can be distinguished in IFN-treated L cells, and two of them appear not to involve the 2-5A synthetase/RNase L pathway.
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Proteolytic processing of a Murray Valley encephalitis virus non-structural polyprotein segment containing the viral proteinase: accumulation of a NS3-4A precursor which requires mature NS3 for efficient processing
More LessThe proteolytic processing of a non-structural polyprotein segment from the cytoplasmic domain of NS2A to the C terminus of NS5 of Murray Valley encephalitis (MVE) virus was examined, when expressed from cDNA via a vaccinia virus recombinant, in transiently transfected COS cells, or synthesized by cell-free translation. Cleavages mediated by the virus-encoded proteinase domain in NS3 at the junctions of NS2A-2B, NS2B-3 and NS4B-5 were catalysed efficiently. However, the cleavage at the NS3-4A junction, also mediated by the NS3 proteinase, was greatly delayed. Little or no NS3 was found, but an 85K precursor molecule accumulated; this was identified as NS3-4A. Termination codons were introduced by site-directed mutagenesis at the junctions of the NS3-4A, NS4A-4B and NS4B-5 genes to generate C-terminal truncations of the MVE virus polyprotein segment. In expression studies of these constructs the predicted NS3-mediated proteolytic cleavages were catalysed, except for that at the NS3-4A junction. In co-infections and co-transfections with constructs encoding the MVE virus non-structural polyprotein region truncated at the C termini of NS3 or NS4A, efficient processing at the NS3-4A site was induced. Thus it appears that the MVE virus polyprotein is cleaved inefficiently in cis at the NS3-4A junction, whereas the site is processed efficiently in trans by mature NS3. The NS3-4A precursor is also seen in flavivirus-infected cells. Its function remains to be determined, but it could play a role in the replication of flavivirus, in view of the importance of polyprotein processing in the regulation of gene expression of positive-stranded RNA viruses, the modulation of processing at the NS3-4A site by NS3 or NS3-containing precursors described in the present study and the importance of NS3 as an integral part of the viral polymerase complex.
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Expression and characterization of glycoprotein gp35 of hepatitis C virus using recombinant vaccinia virus
Complementary DNA clones corresponding to one of the putative structural regions of the hepatitis C virus (HCV) genome were obtained from sera of non-A non-B hepatitis patients. The putative envelope gene was expressed by using a recombinant vaccinia virus carrying this region of the HCV genome. In cells infected with the recombinant vaccinia virus, a glycosylated protein with an M r of about 35K (gp35) was specifically detected by convalescent sera from hepatitis C patients. The sera from rabbits immunized with this recombinant vaccinia virus reacted to the gp35 produced in insect cells and also to gp35 which was translated in vitro in the glycosylated and processed form. The gp35 was used to detect antibodies in sera of only 7 to 23% of HCV patients at various stages of HCV disease. These results suggest that the gp35 of HCV may not have high antigenicity in humans.
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Identification of four VP4 serological types (P serotypes) of bovine rotavirus using viral reassortants
More LessA series of five reassortant viruses each containing the VP4 gene of a distinct bovine rotavirus and the VP7 gene of human rotavirus strain ST3 was prepared, and antisera to these were produced in rabbits. In neutralization tests, these antisera allowed the differentiation of the five original strains (from three different VP7 or G serotypes) into three or possibly four VP4 or P serotypes. All of a further seven bovine rotavirus strains adapted to cell culture were successfully typed by these antisera. There was a degree of cross-reaction between antiserum to the fourth bovine rotavirus P serotype and the predominant human rotavirus serotype. However, antisera raised in guinea-pigs to recombinant VP4 from this serotype showed the bovine serotype to be distinct. There was no significant serological relationship between these four bovine rotavirus P serotypes and previously described P serotypes from rotaviruses isolated from man and non-bovine animals. The predominant bovine rotavirus VP7 serotypes G6 and G10 tended to have distinct P serotypes also.
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Analysis by polymerase chain reaction of the physical state of human papillomavirus type 16 DNA in cervical preneoplastic and neoplastic lesions
More LessIntegration of human papillomavirus (HPV) DNA into the host cell genome is believed to be essential for malignant progression. However unambiguous detection of the physical state of HPV is a difficult and time-consuming procedure. To resolve this issue a simple, rapid and highly sensitive technique of polymerase chain reaction (PCR) has been utilized for detecting the physical state of HPV-16 DNA. Investigations were carried out in 122 cervical specimens comprising the whole spectrum of cervical lesions starting from cervical dysplasia to invasive carcinoma including HPV-16-positive normal controls. A pair of oligonucleotide primers specific to the E2 open reading frame, which is often deleted or disrupted following HPV integration, was used for the study. Distinction between episomal and integrated forms of viral DNA was accomplished by detecting amplification of the E2-specific fragment (1139 bp) in the PCR product. The PCR results were compared with those obtained by the conventional methods of Southern blotting, two-dimensional gel electrophoresis and chromosomal in situ hybridization; a high degree of agreement was observed between the methods. The findings indicate that although integrated forms of HPV-16 DNA were detected in more than 70% of cervical cancer specimens, integration was less frequent (23%) in severe dysplasia and carcinoma in situ. Only 2.5% of cases showed both episomal and integrated forms of HPV-16 DNA. The difference between episomal and integrated forms was statistically significant (P < 0.01). The absence of integration in about 30% of cancer cases suggests that integration of HPV may not be necessary for malignant progression and alternative mechanism(s) of malignant transformation may occur without HPV integration. The PCR test thus provides an effective complement to Southern blotting and two-dimensional gel electrophoresis for accurate detection of the integration of HPV DNA.
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Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera
Existing assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens. These methods have had limited success in the detection of antibodies against the E6 protein. To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells. Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands. This protein reacted in Western blots with an anti-serum raised against a purified E6 fusion protein produced in Escherichia coli. This antiserum, and several others raised against E. coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs). However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target. A RIPA was developed for the detection of anti-E6 protein antibodies in human sera. The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins. The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method. These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.
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Virological and pathological features of mice infected with murine gammaherpesvirus 68
More LessThe primary infection of BALB/c mice with murine herpesvirus 68 (MHV-68) was investigated. When the virus was introduced intranasally, the lung was the main tissue infected, the virus being associated with alveolar epithelium and mononuclear cells. A productive infection lasted for 10 days, after which viral DNA could be detected by in situ hybridization up to 30 days after infection. At that time lymphoproliferative accumulations were also observed in the lung, with formation of germinal centres. Virus could also be recovered from the heart, kidney, adrenal gland and spleen during the primary infection. In addition, the spleen appeared to be the major site of virus persistence, with latently infected cells detected up to 90 days post-infection. During the primary infection, there was atrophy of the thymus and spleen of clinically sick animals. In contrast, lymphoproliferative responses, typified by splenomegaly, were frequently seen in asymptomatic animals. The pattern of infection observed in MHV-68-infected mice is similar to that seen in infectious mononucleosis of man following Epstein–Barr virus infection. The model described in this paper may prove to be useful in studying natural gamma-herpesvirus infections of man and domestic animals.
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Vaccination by cholera toxin conjugated to a herpes simplex virus type 2 glycoprotein D peptide
More LessImmunization of BALB/cJ mice with a peptide corresponding to residues 1 to 23 of glycoprotein D [gD(1–23)] from herpes simplex virus type 2 (HSV-2) elicits antibody responses which correlate with protection against lethal HSV-2 infection. In the present study, we examined the ability of cholera toxin (CTX) to act as an immunogenic carrier for gD(1–23). The number of gD(1–23) residues conjugated to CTX affected its binding to GM1 ganglioside and physiological toxicity, both of which are factors affecting oral immunogenicity. The antibody response elicited after intraperitoneal (i.p.) immunization with the CTX-gD(1–23) conjugate was protective against a lethal i.p. challenge with HSV-2. In other experiments, mice were immunized i.p. on day 0 and subsequent immunizations conducted on days 14 and 28 were administered either intragastrically or intravaginally (i.vag.). Intraperitoneal priming followed by either i.p. or intragastric boosting resulted in anti-HSV-2 antibodies in vaginal washings and in protection against a lethal i.vag. challenge with HSV-2. Intraperitoneal priming followed by i.vag. boosting did not elicit anti-HSV-2 antibodies in vaginal washings and did not protect mice against a lethal i.vag. challenge with HSV-2. These results suggest that CTX can act as a systemic and an oral delivery molecule for the covalently linked gD(1–23) peptide and that such conjugates can elicit protective immune responses against systemic and genital HSV-2 infection.
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