Papers by Carlo De Giuli Morghen
PLOS ONE, Jan 31, 2018
A complete eradication of an HIV infection has never been achieved by vaccination and the search ... more A complete eradication of an HIV infection has never been achieved by vaccination and the search for new immunogens that can induce long-lasting protective responses is ongoing. Avipoxvirus recombinants are host-restricted for replication to avian species and they do not have the undesired side effects induced by vaccinia recombinants. In particular, Fowlpox (FP) recombinants can express transgenes over long periods and can induce protective immunity in mammals, mainly due to CD4-dependent CD8 + T cells. In this context, the class II transactivator (CIITA) has a pivotal role in triggering the adaptive immune response through induction of the expression of class-II major histocompatibility complex molecule (MHC-II), that can present antigens to CD4 + T helper cells. Here, we report on construction of novel FPgp and FPenv recombinants that express the highly immunogenic SIV Gag-pro and Env structural antigens. Several FP-based recombinants, with single or dual genes, were also developed that express CIITA, driven from H6 or SP promoters. These recombinants were used to infect CEF and Vero cells in vitro and determine transgene expression, which was evaluated by real-time PCR and Western blotting. Subcellular localisation of the different proteins was evaluated by confocal microscopy, whereas HLA-DR or MHC-II expression was measured by flow cytometry. Fowlpox recombinants were also used to infect syngeneic T/SA tumour cells, then injected into Balb/c mice to elicit MHC-II immune response and define the presentation of the SIV transgene products in the presence or absence of FPCIITA. Antibodies to Env were measured by ELISA. Our data show that the H6 promoter was more efficient than SP to drive CIITA expression and that CIITA can
Virology, Jul 1, 2003
A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy... more A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy (ART) should be able to replenish virus-specific CD4ϩ T-cells and broaden the virus-specific CD8ϩ T-cell response in order to maintain CD8ϩ T-cell function and minimize viral immune escape after ART cessation. Because a combination of DNA and recombinant poxvirus vaccine modalities induces high levels of virus-specific CD4ϩ T-cell response and broadens the cytolytic activity in naive macaques, we investigated whether the same results could be obtained in SIVmac251-infected macaques. The macaques studied here were long-term nonprogressors that naturally contained viremia but were nevertheless treated with a combination of antiviral drugs to assess more carefully the effect of vaccination in the context of ART. The combination of a DNA expressing the gag and pol genes (DNA-SIV-gp) of SIVmac239 followed by a recombinant fowlpox expressing the same SIVmac genes (FP-SIV-gp) was significantly more immunogenic than two immunizations of FP-SIV-gp in SIVmac251-infected macaques treated with ART. The DNA/FP combination significantly expanded and broadened Gag-specific T-cell responses measured by tetramer staining, ELISPOT, and intracellular cytokine staining and measurement of ex vivo cytolytic function. Importantly, the combination of these vaccine modalities also induced a sizeable expansion in most macaques of Gag-specific CD8-(CD4ϩ) T-cells able to produce TNF-␣. Hopefully, this modality of vaccine combination may be useful in the clinical management of HIV-1-infected individuals.
Antiviral Research, Oct 1, 2010
Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox vi... more Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox virus (FP) represent efficient and safe vaccine vectors, as they correctly express transgenes in human cells, elicit complete immune responses, and show protective efficacy in preclinical animal models. At present, no information is available on the differences in the abortive replication of these two avipox viruses in mammalian cells. In the present study, the replicative cycles of CP and FP, wild-type and recombinants, are compared in permissive and non-permissive cells, using transmission electron microscopy. We demonstrate that in non-permissive cells, the replicative cycle is more advanced in FP than in CP, that human cells, whether immune or not, are less permissive to avipox replication than monkey cells, and that the presence of virus-like particles only occurs after FP infection. Overall, these data suggest that the use of FP recombinants is more appropriate than the use of CP for eliciting an immune response.
PubMed, 1985
Human choroid (HC) cells transformed by Simian virus 40 (HC/SV40) and some of their soft agar-der... more Human choroid (HC) cells transformed by Simian virus 40 (HC/SV40) and some of their soft agar-derived clones were utilized to study the influence of SV40 transformation on the expression of some differentiation parameters. The presence of receptors for DNA and RNA viruses, known to induce cytopathic effects in target cells, always occurred in normal HF and HC/SV40 cells as well as in their derived clones, whereas differences were observed in the expression of influenza A and Sindbis virus receptors. Immunofluorescence assays showed an increase in the expression of class I (HLA-A/B/C) and class II (DR) histocompatibility antigens in hyperdiploid clones compared with both the hypodiploid clones and the normal human cell counterpart. The two surface glycoproteins, fibronectin and laminin, appear only partially correlated with the transformed phenotype of the cells both at optical (immunofluorescence) and supraoptical (immunoelectron microscopy) level. This study confirms the SV40 transformation induces in human choroid cells a modulation of the considered differentiation parameters and that this may be due to a different insertion mode of the SV40 genome in HC cells.
PubMed, Jul 1, 1984
Several clones were isolated from a Simian Virus 40-transformed human choroid cell line (HC/SV40)... more Several clones were isolated from a Simian Virus 40-transformed human choroid cell line (HC/SV40) by the soft agar plating technique. Five of them which showed different morphology, growth capacity and cell density, were plated in monolayer and subjected to ultrastructural, immunological and cytogenetic analyses. The morphological features at both TEM and SEM were distinct for every examined clone but uniform in the cells of the same clone. In particular clone 3 (HC/SV40-C13) showed a greater increase of intracytoplasmic granules, a marker of all cells of choroid origin, while clone 2 (HC/SV40-C12) was totally devoid of them. No virus production was observed at the electron microscope. Immunofluorescence experiments revealed 100% positivity for nuclear T-antigen in all the clones, thus demonstrating the persistence of SV40 genome in these cells. Cytogenetic analysis showed a hyperdiploid number of chromosomes in clones 1 and 3, while clones 2 and 4 were clearly hypodiploid. All clones showed a general tendency to cytogenetic stabilization as compared to the karyotypes found in HC/SV40 parental cell line.
Virus Research, Dec 1, 2013
The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully e... more The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected.
Journal of Translational Medicine, Apr 21, 2010
Background: Around half million new cases of cervical cancer arise each year, making the developm... more Background: Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPVrelated precancerous lesions. Methods: Three different immunisation regimens were tested in a pre-clinical trial in rabbits to evaluate the humoral and cell-mediated responses of a putative HPV-16 vaccine. Fowlpoxvirus (FP) recombinants separately expressing the HPV-16 E6 (FP E6) and E7 (FP E7) transgenes were used for priming, followed by E7 protein boosting. Results: All of the protocols were effective in eliciting a high antibody response. This was also confirmed by interleukin-4 production, which increased after simultaneous priming with both FP E6 and FP E7 and after E7 protein boost. A cell-mediated immune response was also detected in most of the animals. Conclusion: These results establish a preliminary profile for the therapy with the combined use of avipox recombinants, which may represent safer immunogens than vaccinia-based vectors in immuno-compromised individuals, as they express the transgenes in most mammalian cells in the absence of a productive replication.
Journal of Biotechnology, Aug 1, 1997
Synthetic oligonucleotides corresponding to specific V 3 loop portions of two HIV-1 isolates, SC ... more Synthetic oligonucleotides corresponding to specific V 3 loop portions of two HIV-1 isolates, SC and WMJ2, were expressed in the flagella of a Salmonella live-vaccine strain. Expression of the inserted epitopes in flagellin and their exposure at the surface of flagellar filaments were shown by immunoblotting and immunogold labeling with anti-flagellin (Salmonella d) and anti-HIV-1(IIIB) V 3 loop peptide sera. Live recombinant Salmonella strains expressing either one of the two V 3 loop inserts were administered intraperitoneally to BALB/c mice. All these animals developed antibodies specific for the heterologous glycoprotein 120 (gp120) of HIV-1 MN strain, as detected by enzyme-linked immunosorbent assays (ELISA); two of the sera had neutralizing activity against the heterologous HIV-1 MN strain. Moreover, oral administration of the live Salmonella recombinant strains to mice evoked specific IgA directed against gp120.
Vaccine, May 1, 2003
Three different prime-boost immunization protocols were tested in rabbits and their immune respon... more Three different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of identifying a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic chimera simian/human immunodeficiency virus (SHIV) 89.6P. Protocols were based on priming with two fowlpox (FP) recombinant vectors and two expression plasmids, which express either the simian immunodeficiency virus (SIV)mac 239 gag/pol or the human immunodeficiency virus (HIV-1)env 89.6P genes, followed by boosting with virus-like particles (VLP). All protocols were effective in eliciting homologous neutralizing Ab and highlighted the efficacy of VLP boosting. The FP vector was less efficient than plasmid DNA in inducing Ab against the gag core proteins. Analysis of cytokine expression 5 months after last immunization indicated that priming with pcDNA3gag/pol SIV and FPenv 89.6P followed by VLP boosting generated a T helper (Th0) profile and a good Ab titer, suggesting a potential protocol to be tested in the SHIV-macaque model of HIV-1 infection.
Virology Journal, Mar 4, 2021
Background: Zika virus (ZIKV) has been declared a public health emergency that requires developme... more Background: Zika virus (ZIKV) has been declared a public health emergency that requires development of an effective vaccine, as it might represent an international threat. Methods: Here, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine candidates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verified for correct transgene expression by Western blotting and by immunofluorescence in different cell lines. The production of virus-like particles as a result of env gene expression was also demonstrated by electron microscopy. BALB/c mice were immunosuppressed with dexamethasone and immunized following a prime-boost strategy in a heterologous protocol where pVAXzenv was followed by FPzenv, to evaluate the immunogenicity of the Env protein. The mice underwent a challenge with an epidemic ZIKV after the last boost. Results: These data show that the ZIKV Env protein was correctly expressed in both normal human lung fibroblasts (MRC-5 cells) and green monkey kidney (Vero) cells infected with FPzenv, and that the transgene expression lasted for more than 2 weeks. After mucosal administration of FPzenv, the immunized mice showed specific and significantly higher humoral responses compared to the control mice. However, virus neutralizing antibodies were not detected using plaque reduction assays. Conclusions: Although BALB/c mice appear to be an adequate model for ZIKV infection, as it mimics the natural mild infection in human beings, inadequate immune suppression seemed to occur by dexamethasone and different immune suppression strategies should be applied before challenge to reveal any protection of the mice.
Various derivatives of trienomycin A (1) were prepared and tested for cytocidal activities. All t... more Various derivatives of trienomycin A (1) were prepared and tested for cytocidal activities. All the derivatives except for 22-O-methyltrienomycin A (5) showed reduced cytotoxicity compared with 1. It is concluded that the existence of a triene moiety, free 13-OHand an acyl group at C-l l owe important role for cytocidal activity.
Annals of Microbiology, 2004
The efficiency of a decontamination procedure by sonication for different dental instruments afte... more The efficiency of a decontamination procedure by sonication for different dental instruments after experimental microbial and viral contamination was tested. Both germicidal and virucidal activity of sonication in the presence or absence of a cationic biobiguanid disinfectant was assessed following three different disinfection/sterilisation protocols. Dental instruments were contaminated with a mixed culture of Enterococcus faecium, Staphylococcus sp., Pseudomonas aeruginosa, Mycobacterium sp., Escherichia coli and Bacillus subtilis, or with Poliovirus type 1 and Herpesvirus simplex type 1 (HHV-1), exposed to ultrasonic treatment in an ultrasonic bath and the surviving microorganisms titered. The results showed that an effective disinfection of dental instruments, expressed by an equal or higher than 4 logs microbial and viral reduction, can be obtained after 15 min or 10 min sonication in an ultrasonic cleaner equipped with a Sweep System Technology. Conversely, by the combined action of chemical disinfection and ultrasonic treatment in the same device, a sterilising effect was obtained after only 5 min for microbial and 10-15 min for virally contaminated instruments. The synergistic effect of chemical and physical means, as already accepted as an effective cleaning procedure of medical instruments, can therefore be applied to obtain a safe and effective sterilisation of dental instruments potentially contaminated by organic fluids and dental material harbouring pathogenic microbes and viruses.
Journal of Virological Methods, 2013
The development of an effective prophylactic vaccine is still necessary to improve the safety of ... more The development of an effective prophylactic vaccine is still necessary to improve the safety of the conventional although-discontinued smallpox vaccine, and to protect from the threat of deliberate release of variola virus. This need also arises from the number of new cases of animal orthopoxvirus infections each year, and to reduce the risk to animal handlers. Fowlpox (FP) recombinants only replicate in avian species and have been developed against human infectious diseases, as they can elicit an effective immune response, are not cross-reactive immunologically with vaccinia, and represent safer and more promising immunogens for immunocompromised individuals. The aim of this study was the characterisation of two new fowlpox recombinants expressing the A33R vaccinia virus gene either alone (FP A33R) or with the green fluorescent protein (FP A33R-GFP) to verify whether GFP can affect the expression of the transgene. The results show that both FP A33R and FP A33R-GFP can express A33R correctly, but A33R mRNA and protein synthesis are higher by FP A33R than by FP A33R-GFP. Therefore, GFP co-expression does not prevent, but can reduce the level of a vaccine protein, and may affect the protective efficacy of the immune response.
Journal of Virological Methods, Jun 1, 2009
Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype and the expressio... more Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype and the expression of the E6 and E7 proteins, which can bind to the p53 and p105Rb host cell-cycle regulatory proteins, is related to its tumorigenicity. Virus-like-particle (VLP)-based immunogens developed recently are successful as prophylactic HPV vaccines. However, given the high number of individuals infected already with HPV and the absence of expression of the L1 structural protein in HPV-infected or HPV-transformed cells, an efficient therapeutic vaccine targeting the non-structural E6 and E7 oncoproteins is required. In this study, two new fowlpox virus (FPV) recombinants encoding the HPV-16 E6 and E7 proteins were engineered and evaluated for their correct expression in vitro, with the final aim of developing a therapeutic vaccine against HPV-related cervical tumors. Although vaccinia viruses expressing the HPV-16 and HPV-18 E6 and E7 oncoproteins have already been studied, due to their natural host-range restriction to avian species and their ability to elicit a complete immune response, FPV recombinants may represent efficient and safer vectors also for immunocompromised hosts. The results indicate that FPV recombinants can express correctly the E6 and E7 oncoproteins, and they should represent appropriate vectors for the expression of these oncoproteins in human cells.
PLOS ONE, Jan 31, 2018
A complete eradication of an HIV infection has never been achieved by vaccination and the search ... more A complete eradication of an HIV infection has never been achieved by vaccination and the search for new immunogens that can induce long-lasting protective responses is ongoing. Avipoxvirus recombinants are host-restricted for replication to avian species and they do not have the undesired side effects induced by vaccinia recombinants. In particular, Fowlpox (FP) recombinants can express transgenes over long periods and can induce protective immunity in mammals, mainly due to CD4-dependent CD8 + T cells. In this context, the class II transactivator (CIITA) has a pivotal role in triggering the adaptive immune response through induction of the expression of class-II major histocompatibility complex molecule (MHC-II), that can present antigens to CD4 + T helper cells. Here, we report on construction of novel FPgp and FPenv recombinants that express the highly immunogenic SIV Gag-pro and Env structural antigens. Several FP-based recombinants, with single or dual genes, were also developed that express CIITA, driven from H6 or SP promoters. These recombinants were used to infect CEF and Vero cells in vitro and determine transgene expression, which was evaluated by real-time PCR and Western blotting. Subcellular localisation of the different proteins was evaluated by confocal microscopy, whereas HLA-DR or MHC-II expression was measured by flow cytometry. Fowlpox recombinants were also used to infect syngeneic T/SA tumour cells, then injected into Balb/c mice to elicit MHC-II immune response and define the presentation of the SIV transgene products in the presence or absence of FPCIITA. Antibodies to Env were measured by ELISA. Our data show that the H6 promoter was more efficient than SP to drive CIITA expression and that CIITA can
Virology, Jul 1, 2003
A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy... more A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy (ART) should be able to replenish virus-specific CD4ϩ T-cells and broaden the virus-specific CD8ϩ T-cell response in order to maintain CD8ϩ T-cell function and minimize viral immune escape after ART cessation. Because a combination of DNA and recombinant poxvirus vaccine modalities induces high levels of virus-specific CD4ϩ T-cell response and broadens the cytolytic activity in naive macaques, we investigated whether the same results could be obtained in SIVmac251-infected macaques. The macaques studied here were long-term nonprogressors that naturally contained viremia but were nevertheless treated with a combination of antiviral drugs to assess more carefully the effect of vaccination in the context of ART. The combination of a DNA expressing the gag and pol genes (DNA-SIV-gp) of SIVmac239 followed by a recombinant fowlpox expressing the same SIVmac genes (FP-SIV-gp) was significantly more immunogenic than two immunizations of FP-SIV-gp in SIVmac251-infected macaques treated with ART. The DNA/FP combination significantly expanded and broadened Gag-specific T-cell responses measured by tetramer staining, ELISPOT, and intracellular cytokine staining and measurement of ex vivo cytolytic function. Importantly, the combination of these vaccine modalities also induced a sizeable expansion in most macaques of Gag-specific CD8-(CD4ϩ) T-cells able to produce TNF-␣. Hopefully, this modality of vaccine combination may be useful in the clinical management of HIV-1-infected individuals.
Antiviral Research, Oct 1, 2010
Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox vi... more Due to their natural host-range restriction to avian species, canarypox virus (CP) and fowlpox virus (FP) represent efficient and safe vaccine vectors, as they correctly express transgenes in human cells, elicit complete immune responses, and show protective efficacy in preclinical animal models. At present, no information is available on the differences in the abortive replication of these two avipox viruses in mammalian cells. In the present study, the replicative cycles of CP and FP, wild-type and recombinants, are compared in permissive and non-permissive cells, using transmission electron microscopy. We demonstrate that in non-permissive cells, the replicative cycle is more advanced in FP than in CP, that human cells, whether immune or not, are less permissive to avipox replication than monkey cells, and that the presence of virus-like particles only occurs after FP infection. Overall, these data suggest that the use of FP recombinants is more appropriate than the use of CP for eliciting an immune response.
PubMed, 1985
Human choroid (HC) cells transformed by Simian virus 40 (HC/SV40) and some of their soft agar-der... more Human choroid (HC) cells transformed by Simian virus 40 (HC/SV40) and some of their soft agar-derived clones were utilized to study the influence of SV40 transformation on the expression of some differentiation parameters. The presence of receptors for DNA and RNA viruses, known to induce cytopathic effects in target cells, always occurred in normal HF and HC/SV40 cells as well as in their derived clones, whereas differences were observed in the expression of influenza A and Sindbis virus receptors. Immunofluorescence assays showed an increase in the expression of class I (HLA-A/B/C) and class II (DR) histocompatibility antigens in hyperdiploid clones compared with both the hypodiploid clones and the normal human cell counterpart. The two surface glycoproteins, fibronectin and laminin, appear only partially correlated with the transformed phenotype of the cells both at optical (immunofluorescence) and supraoptical (immunoelectron microscopy) level. This study confirms the SV40 transformation induces in human choroid cells a modulation of the considered differentiation parameters and that this may be due to a different insertion mode of the SV40 genome in HC cells.
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Papers by Carlo De Giuli Morghen