Immune responses induced by replication-defective adenovirus expressing the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine, causing significant economic losses to swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of this disease. The goal of this study was to design and evaluate a replication-defective recombinant adenovirus, rAdP97c, expressing the C-terminal portion of P97 adhesin (P97c), an important pathogenesis-associated protein of M. hyopneumoniae, as a new vaccine candidate against M. hyopneumoniae infection. P97c-specific immune responses were evaluated in BALB/c mice following intranasal and intramuscular inoculation with rAdP97c. Mice inoculated by both routes of immunization produced significant levels of specific immunoglobulin G (IgG) antibodies in the serum and in bronchoalveolar lavage fluids (BALs). Animals immunized intranasally also produced a significant level of P97c-specific IgA in BALs. Intramuscular inoculation of rAdP97c induced a systemic and mucosal Th1-type biased response, evidenced by the predominance of IgG2a in the serum and BALs, whereas intranasal inoculation resulted in a mixed Th1/Th2-type response (balanced levels of IgG1 and IgG2a) in both sytemic and mucosal compartments. P97c-specific antibodies were able to inhibit the growth of M. hyopneumoniae cells in vitro. These data suggest that rAdP97c vaccine may represent a new strategy for controlling infection by M. hyopneumoniae.

Determinants of low-risk and high-risk cervical human papillomavirus infections in Montreal University students.

BACKGROUND: Previous studies have been inconsistent about the degree of sexual transmissibility of cervical human papillomavirus (HPV) infection. The authors hypothesize that risk factors for HPV infection vary according to HPV type. GOAL: To estimate the prevalence of HPV infection in asymptomatic women and to identify risk factors for overall HPV infection and HPV infection by oncogenic and nononcogenic type. STUDY DESIGN: A cross-sectional survey was conducted at the McGill University clinic in Montreal. Cervical specimens were collected from 489 female students presenting at the clinic for a routine Papanicolaou test. Data on potential risk factors was obtained by questionnaire. Human papillomavirus DNA was detected by the polymerase chain reaction using consensus primers (MY09/11) followed by hybridization with generic and type-specific probes using Southern blot and dot blot techniques. RESULTS: The overall HPV prevalence was 21.8%. A low-risk HPV infection was found in 6.2% of the women, 11.8% had a high-risk HPV infection (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58), 7.1% had an unknown HPV type, and 2.7% had a multiple type infection. Two profiles emerged for sexual activity and risk of HPV infection according to oncogenic risk after multivariate analysis. Lifetime frequency of sexual intercourse and lifetime number of oral sex partners was associated with high-oncogenic-risk HPV infections; however, HPV infection with low-oncogenic-risk types was invariant with respect to markers of sexual activity. CONCLUSION: These results suggest that there are differences in epidemiologic correlates of transmission between low-risk and high-oncogenic-risk HPV types based on oncogenicity. This finding has important implications for primary prevention of HPV infection and cervical cancer precursors.

T-cell receptor-mediated anergy of a human immunodeficiency virus (HIV) gp120-specific CD4(+) cytotoxic T-cell clone, induced by a natural HIV type 1 variant peptide.

Human immunodeficiency virus type 1 (HIV-1) infection triggers a cytotoxic T-lymphocyte (CTL) response mediated by CD8(+) and perhaps CD4(+) CTLs. The mechanisms by which HIV-1 escapes from this CTL response are only beginning to be understood. However, it is already clear that the extreme genetic variability of the virus is a major contributing factor. Because of the well-known ability of altered peptide ligands (APL) to induce a T-cell receptor (TCR)-mediated anergic state in CD4(+) helper T cells, we investigated the effects of HIV-1 sequence variations on the proliferation and cytotoxic activation of a human CD4(+) CTL clone (Een217) specific for an epitope composed of amino acids 410 to 429 of HIV-1 gp120. We report that a natural variant of this epitope induced a functional anergic state rendering the T cells unable to respond to their antigenic ligand and preventing the proliferation and cytotoxic activation normally induced by the original antigenic peptide. Furthermore, the stimulation of Een217 cells with this APL generated altered TCR-proximal signaling events that have been associated with the induction of T-cell anergy in CD4(+) T cells. Importantly, the APL-induced anergic state of the Een217 T cells could be prevented by the addition of interleukin 2, which restored their ability to respond to their nominal antigen. Our data therefore suggest that HIV-1 variants can induce a state of anergy in HIV-specific CD4(+) CTLs. Such a mechanism may allow a viral variant to not only escape the CTL response but also facilitate the persistence of other viral strains that may otherwise be recognized and eliminated by HIV-specific CTLs.

Choristoneura fumiferana granulovirus: sequence analysis and 5' characterization of ORF891.

A gene located immediately upstream of the granulin gene of Choristoneura fumiferana (ChfuGV) granulovirus was identified, sequenced and named ORF891. The determined, putative open reading frame (ORF) of 891 bp encodes an estimated 34.6 kDa protein. The 5' end transcript of the gene was mapped and analysed. A putative promoter region organization of ChfuGV ORF891 contains a consensus late baculovirus promoter element, TAAG, and two putative early TATA boxes similar to the promoters of ORF909 of Cryptophlebia leucotreta granulovirus (ClGV). Sequence comparisons of ChfuGV ORF891 with ClGV ORF909 and Cydia pomonella granulovirus (CpGV) ORF124R showed respective homologies of 60.9 and 63.9% for nucleotides and 46.3% and 49.3% for amino acids. Homology of ChfuGV ORF891 with ME53 ORF of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was 68.2% for nucleotides but a total lack of homology for amino acid sequences. Two zinc finger motifs are also associated with ChfuGV ORF891.

Human papillomavirus and prognoses of patients with cancers of the upper aerodigestive tract.

BACKGROUND: Some studies have shown that human papillomavirus (HPV) infection may play not only an etiologic role in anogenital cancers but also a role in the clinical outcome. The objective of the current study was to determine whether detection of HPV DNA in primary squamous cell carcinomas of the upper aerodigestive tract (UADT) is a prognostic factor in patients with the disease. METHODS: The authors analyzed archival specimens of UADT tumors from 101 randomly selected patients with evaluable samples for HPV DNA detection. HPV testing was performed using a general primer-mediated polymerase chain reaction. RESULTS: The overall detection rate was 16.8% (17 of 101 specimens). HPV DNA was detected at higher rates in specimens from younger patients and in well-differentiated tumors. Pharyngeal tumors were more likely to be HPV positive (30.0%) than buccal (10.3%) or laryngeal tumors (15.4%), but the differences were not significant. The detection rate was similar for T1-T2 tumors (17.4%) and T3-T4 tumors (15.6%). However, tumors without lymph node metastasis were more likely to be HPV positive (21.4%) than tumors with lymph node involvement (6.5%). Kaplan-Meier and Cox regression survival analyses did not show any difference in overall or disease free survival according to HPV detection. CONCLUSIONS: Although the HPV DNA detection rate was slightly higher in local than in regionally spread tumors, our results support the hypothesis that it is very unlikely that HPV detection plays any role in the prognoses of patients with UADT squamous cell carcinoma.

Sequence analysis and expression of the polyhedrin gene of Choristoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV).

The segmented double-stranded RNA genome of Choristoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV) was extracted, polyadenylated, reverse-transcribed into cDNA and cloned. The cDNA clones that hybridized to the smallest genomic segment (segment 10) were identified, and its nucleotide sequence was determined. Genome segment 10 of CfCPV was found to be 1171 nucleotides in length with a single open reading frame in one strand capable of coding a predicted protein of 258 residues (Mr of 29,795), consistent with an apparent Mr of 30.5 kDa determined by SDS-PAGE of purified polyhedrin. Comparison of the nucleotide and amino acid sequences of the polyhedrin gene of CfCPV with those of other CPVs and with several nuclear polyhedrosis viruses revealed no particular homology. Analysis of the hydrophilic profiles and predicted secondary structures of Bombyx mori (BmCPV), Euxoa scandens (EsCPV) and CfCPV indicated the presence of seven similar regions located at the amino terminus of the polyhedrin polypeptide of the three viruses. The expression of the cloned CfCPV polyhedrin gene in Escherichia coli demonstrated that this polyhedrin has the property of self-assembly, since the production of crystal-like occlusion with a well-defined crystalline lattice structure was observed.

Immunogenicity of E. coli-expressed sigma3 protein of avian reovirus in chickens.

An E. coli-expressed fusion protein was used to study the immunogenicity of avian reovirus sigma3 protein in chickens. The protein induced the production of specific antibodies detectable by immunofluorescence, ELISA and immunoblot. The antibody titre was low as determined by ELISA and was negative by virus neutralization test. However, when tested in passive immunization studies, the chicken antiserum specific to this protein and control antiserum from chickens vaccinated with avian reovirus SI 133 strain showed some protection against virus-induced mortality in 1-day-old chicks. The results thus confirm the importance of humoral immunity against avian reovirus infection and indicate that sigma3 protein may play a role in the induction of protective antibodies.

Identification and sequence analyses of the granulin gene of Choristoneura fumiferana granulovirus.

The nucleotide sequence of the granulin gene of Choristoneura fumiferana granulovirus (CfGV) was determined. The gene encodes a protein of 248 amino acids with a predicted Mr of 29.299 kDa. The granulin genes of Trichoplusia ni, Pieris brassicae and Cryptophlebia leucotreta granuloviruses showed homologies ranging from 76.7-80.5% for nucleotide sequences and 84.2-88.3% for amino acid sequences when compared to CfGV. The secondary structure of CfGV granulin protein, including the hydrophilic (polar) and hydrophobic (basic) regions, was predicted and found to be similar to other granulins. A very late baculovirus promoter motif, ATAAG, was found within the putative promoter region of the CfGV granulin gene.

Evidence for the multimeric nature and cell binding ability of avian reovirus sigma 3 protein.

It has been suggested that avian reovirus sigma 3 protein is analogous to sigma 1 trimer, the mammalian reovirus attachment protein. We have investigated the multimeric nature and cell binding ability of sigma 3 protein. The data presented here demonstrate that sigma 3 protein is a multimer in its undisrupted form as determined by SDS-PAGE in non-dissociating conditions. However, virion-associated sigma 3 protein and COS-7 cell-expressed protein behaved differently in SDS-PAGE, suggesting a need for virus-associated factor(s) to control the multimerization of the protein. The data also show that Escherichia coli expressed sigma 3 fusion protein (sigma 3-MBP) in its multimeric form is capable of attaching to Vero cells. The binding was found to be specific and receptor mediated by the fact that it was inhibited by a monoclonal antibody specific for sigma 3 protein and by competition with avian reovirus particles. As determined by a reverse experiment, sigma 3-MBP was also able to reduce the virus p.f.u. in monolayer cell cultures, indicating the important role of sigma 3 protein in the initiation of virus infection.

Human papillomavirus DNA in invasive cervical carcinomas and its association with patient survival: a nested case-control study.

This study sought to examine the association between the presence of human papillomavirus (HPV) DNA in invasive cervical cancer and prognosis. A case-control study was undertaken nested in a cohort of 208 patients with invasive cervical carcinoma in Montreal. All 40 deceased patients formed the case groups. A control group of equal size was selected by matching to each case (1:1) a patient of the same age and year of admission who had survived her disease. HPV DNA was detected by the use of a PCR protocol. The odds ratio (OR) for cervical cancer death was computed by logistic regression. Detection of HPV, particularly of types 16 and 18, was negatively correlated with disease stage and histological grade. The OR for death was 0.27 [95% confidence interval (CI), 0.1-0.8] for those whose tumors were positive for HPV DNA versus those in whom HPV DNA was not detected. After adjusting for the confounding effects of stage and grade, the prognostic effect was somewhat reduced, with an OR of 0.34 (CI, 0.1-1.1), which was no longer significant. The magnitude of the HPV-survival association was not altered when the analyses were restricted to carcinomas of stages I and II. Regardless of the mechanism for the prognostic role, detection of HPV DNA in primary tumors may play an important adjunct role in assessing prognosis of patients with invasive cervical cancer.

Characterization of monoclonal antibodies against avian reovirus strain S1133.

Monoclonal antibodies (MAbs) were produced against a vaccinal S1133 strain of avian reovirus. Characterization of six MAbs in Western blotting, radioimmunoprecipitation and gold immunoelectron microscopy revealed that the MAbs were specific to the outer capsid proteins, mu2/mu2c, sigma2 and sigma3. Two of three MAbs, directed against sigma2 protein, neutralized the virus infectivity in a broadly specific manner, whereas the third had no neutralizing activity. The only MAb which reacted with sigma3 protein showed a type-specific neutralizing activity. Two MAbs recognizing the mu2/mu2c proteins failed to neutralize the virus infectivity.

Strain variability and localization of important epitopes on the major structural protein (VP2) of infectious pancreatic necrosis virus.

Infectious pancreatic necrosis virus (IPNV), a birnavirus, is an important pathogen in fish farms. Analyses of viral proteins showed that VP2 is the major structural and immunogenic polypeptide of the virus. All neutralizing monoclonal antibodies (mAbs) against IPNV are specific to VP2 and bind to continuous or discontinuous epitopes. In order to determine which parts of the protein are involved in antigenic variations, five IPNV strains were sequenced over the VP2 coding region. Comparison of the sequences obtained with three previously published strains revealed a central variable domain (positions 183 to 335) which encompasses two hydrophilic hypervariable segments. Viral mutants which escaped neutralization were then selected with anti-VP2 mAbs directed against discontinuous epitopes. Sequencing of three mutants revealed a single amino acid mismatch in each of them. All of these substitutions occurred in the hypervariable segments, suggesting that these regions are involved in the formation of a discontinuous epitope. Finally, expression of different truncated VP2s in Escherichia coli allowed localization of the binding site for neutralizing mAbs which recognize continuous epitopes. One of these mAbs bound to the region adjacent to the C-terminus of the variable domain of VP2, while two others reacted with the central and C-terminal parts of the variable domain. No antibody reacted with the N-terminus of VP2. These results suggest that the variable domain of VP2 and the 20 adjacent amino acids of the conserved C-terminal part are the most important in inducing an immune response for the protection of animals.

Characterization of the small open reading frame on genome segment A of infectious pancreatic necrosis virus.

The genome of infectious pancreatic necrosis virus (IPNV) is composed of two segments of dsRNA. The larger segment contains a small ORF partly overlapping the 5' end of the polyprotein reading frame. Yet very little is known about this possible new gene, which presumably codes for a 17 kDa polypeptide (VP5). The region of the viral genome which encompasses the small ORF was reverse-transcribed and amplified by PCR before cloning and sequencing. Analysis of the sequences obtained from five different virus strains revealed that the small ORF is not found on one of them, and that it is truncated on two others. Moreover, the deduced amino acid sequences did not appear to be well conserved. Despite the large variations between IPNV strains at the genomic level, all predicted VP5 are arginine-rich basic polypeptides. To verify whether the small ORF is translated into protein in fish cells, the 17 kDa polypeptide of the VR-299 strain was expressed as fusion protein in a prokaryotic expression vector and used to produce a specific antiserum. This antiserum reacted with concentrated virus in an immunodot assay indicating that VP5 is synthesized in infected cells, but probably only in small quantities. When tested with 12 other IPNV strains, results were less conclusive than those obtained with strain VR-299. Nevertheless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain lacks the small ORF.

Cloning, sequencing and expression of the S1 gene of avian reovirus.

The S1 genome segment of avian reovirus strain S1133 was cloned and completely sequenced. The sequence comprised 1636 bp with three distinct open reading frames (ORFs), suggesting the gene was polycistronic in nature. The three ORFs from 5' to 3' were predicted to encode polypeptides of 9.8, 3.8 and 34.9 kDa, respectively. Of the three ORFs, only the third possessed the AUG initiation codon in an optimum context for translation. The third ORF-encoded protein, 326 amino acids in length, was expressed in Escherichia coli and used as antigen in immunoblots. The protein was concluded to be sigma 3 on the basis of its recognition by a chicken anti-reovirus antiserum and due to the fact that a mouse antiserum raised against it recognized specifically the viral sigma 3 polypeptide. Sequence comparison of the avian reovirus S1 gene with its mammalian counterpart did not show any significant similarity between the two. However, amino acid sequence analysis and the predicted existence of a heptapeptide repeat pattern, as well as the relatively high frequency of alpha-helix structures in the amino terminal portion of sigma 3 suggests that this protein is structurally, and probably functionally, related to mammalian reovirus sigma 1 protein.

Alterations in cell-cell communication in human papillomavirus type 16 (HPV16) transformed rat myoblasts.

A reduction of gap-junctional intercellular communication (GJIC) often accompanies neoplastic transformation. The present work demonstrates that transformation by the oncogenic human DNA virus, human papilloma virus 16(HPV16), also reduces GJIC between L6 rat myoblasts. HPVs are associated with anogenital cancers, the incidence of which is increasing in HIV positive patients of both sexes. Using videofluorescence imaging of Fura-2 loaded cells a lack of GJIC between transformed HPV16-L6 cells was first indicated by uncoordinated brief [Ca2+]i spikes in clusters of DMSO-treated HPV16-L6 cells instead of the synchronous, sustained [Ca2+]i surges in clusters of DMSO-treated L6 cells. Reduced GJIC between HPV16-L6 cells was demonstrated directly by a much reduced transfer of lucifer yellow dye from HPV16-L6 cells, which had been loaded with the dye through electroporation with an EPIZAP II in situ electroporator, to neighbouring nonelectroporated HPV16-L6 cells. One reason for this reduced GJIC between HPV16-L6 cells could have been their dramatically enhanced activity of membrane-associated PKC which is known to phosphorylate connexins and down-regulate gap junctions. However, the main reason was the viral-induced inhibition of the expression of a major gap junction component, Cx43 (Connexin 43), in the transformed myoblasts.

HIV-1 gp120/160 expressing cells upregulate HIV-1 LTR directed gene expression in a cell line transfected with HIV-1 LTR-reporter gene constructs.

The results described in this paper demonstrate that HIV-1 gp120 can upregulate gene expression directed by the HIV-1 LTR. Briefly, exposing responder CD4+CEM-T4 ID5 cells to stimulator CEMgp120/160 expressing cells (stably transfected with HIV-1 LTR-CAT and HIV-1 gp160, respectively) resulted in the increased synthesis of the CAT enzyme. Control non-transfected CEM-T4 cells did not induce the synthesis of CAT. In addition, when the responder cell line, U937-1C5 which also contains stably transfected HIV-1 LTR-CAT plasmid was exposed to irradiated CEM gp120/160 cells, there was no synthesis of the CAT enzyme. Neither recombinant gp120 nor gp160 were able to stimulate the synthesis of CAT in the responder cells. These results indicate that the mechanism by which gp120/160 expressed on transfected cells increase CAT synthesis in responder cells may be dependent on the manner which the protein is presented in association with accessory molecules. Moreover, recombinant soluble CD4 and anti-CD4 monoclonal antibodies inhibited CEM gp120/160 induced expression of HIV-1 LTR-directed expression in CEM-1D5 cells. Based on these results we hypothesize that HIV or its envelope protein, gp120, upon interaction with its receptor, the CD4 molecule on T helper cells, transduces a signal which translates into the upregulation of the gene expression directed by the HIV-1 LTR.

HIV-1 gp120/160 expressing transfected cell clones to evaluate the antibody-dependent cellular cytotoxicity (ADCC) during the course of HIV-1 infection.

Transfection by electroporation of the CEM.NKR cell line with a plasmid containing the HIVNL43 gp160 gene resulted in the establishment of HIV-1 gp120/160 expressing CEM.NKR cell clones. The cell-surface expression of the recombinant viral envelope is stable and does not lead to syncytium formation among a substantial number of cells expressing envelope glycoprotein, suggesting that these cells are suitable as targets to monitor HIV-specific ADCC activities with the stage or the progression of HIV-1 disease. By using these cells, the ability of sera from HIV-1 seropositive individuals to mediate ADCC against HIV-1 gp120/160 transfected cells in an antigen-specific manner was established. Approximately 63% of the HIV-infected individuals representing all stages of infection have virus-specific ADCC antibodies. Moreover, sera from healthy seropositive (CDC class II) subjects mediated substantially higher levels of ADCC activity (25.7 +/- 11.5%) than did sera from individuals in CDC class III (10.3 +/- 8.9%) or IVC1 (6.4 +/- 8.1%). The mean ADCC activity for the sera from the seronegative volunteers was 2 +/- 1.3%. The correlation between the ADCC level and CD4 cell depletion remains uncertain. Therefore, the established transfected cell line may be used to probe the role of gp120/160-specific ADCC activity in the development of AIDS and may also prove useful in screening human and murine monoclonal antibodies with potential ADCC activity. Such monoclonals may be useful for future immunotherapeutic agents in conjunction with antiviral chemotherapy.

Expression in Escherichia coli of the cloned polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus.

Cloned cDNA of genomic segment 10 of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was placed downstream from the lambda PL promoter in expression plasmid pRC23 and expressed in Escherichia coli cells. A polypeptide of the same molecular weight (28 kDa) as natural polyhedrin was synthesized at the level of approximately 10% of total host cell protein. This polypeptide was identified as CPV polyhedrin (r-polyhedrin) after comparative studies. The r-polyhedrin did not form any crystalline structure in E. coli cells but instead accumulated in the form of an insoluble inclusion body, even though natural polyhedrin is known to form a crystalline matrix (polyhedra) in infected insect cells. The purified r-polyhedrin complex, like natural polyhedra, was not soluble in neutral or acidic buffer but soluble in alkaline buffer. Upon solubilization, the r-polyhedrin complex did not undergo proteolytic degradation, while natural polyhedra were digested into small peptides by the associated protease. Incubation of r-polyhedrin with natural polyhedra in alkaline buffer, however, degraded the r-polyhedrin, resulting in an identical profile of peptide products to that of natural polyhedra. These results indicate that even though r-polyhedrin molecules produced in E. coli cells are not in the natural conformation, the molecules can present the identical cleavage sites to the polyhedra-associated alkaline protease. Experiments showed that the alkaline protease was associated with the matrix of polyhedra and not with virus particles.

Antigenic characterization of serogroup 'A' of infectious pancreatic necrosis virus with three panels of monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against three serotypes of infectious pancreatic necrosis virus (IPNV): A1 (LWVRT 60-1, U.S.A.), A2 (d'Honnincthun, France) and A9 (Jasper, Canada). Each panel of MAbs (identified as LW, HF and JA) was analysed by ELISA with the 10 proposed serotypes of IPNV and their specificity defined by immunoprecipitation and Western immunoblotting analysis. A first group of MAbs, directed against the outer capsid protein VP2, reacted with linear or conformational epitopes. A second group of MAbs, directed against the internal protein VP3, reacted with linear epitopes. There was no relationship between the neutralizing property of anti-VP2 MAb and the configuration of the epitope that it recognized. The MAbs were used for antigenic characterization of serogroup A. Each panel of MAbs showed a characteristic pattern of reactivity. The European HF series was predominantly cross-reactive and detected conserved epitopes among the 10 serotypes for both VP2 and VP3. The North American LW and JA series identified a group of conserved epitopes on VP3 and new specific epitopes on VP2 and VP3. The higher variability observed for VP2 in comparison with VP3 is one example of how external pressures may promote natural selection of those epitopes required for virus survival. Our results are consistent with an ancestral relationship of the European to the North American strains, the latter having developed new antigenic determinants upon evolution in their new geographical location.