Carlo De Giuli Morghen

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

Protective properties of non-nucleoside reverse transcriptase inhibitor (MC1220) incorporated into liposome against intravaginal challenge of Rhesus Macaques with RT-SHIV

In the absence of an effective vaccine against HIV, it is urgent to develop an effective alternative such as a microbicide. Single and repeated applications of MC1220 microbicide were evaluated in macaques. First, animals were given a single application of 0.5% or 1.5% MC1220-containing liposomal gel. A second group were treated with 0.5% MC1220 once a day for 4 days. The control groups were treated by liposomal gel alone. Thirty minutes after the last application, animals were challenged with RT-SHIV. In the first protocol, 2 of 4 animals treated by 0.5% of the MC1220 and 2 of 5 treated by 1.5% were protected. In the second protocol, 3 of 5 treated animals were protected and 5 of 5 controls were infected. The RNA viral load at necropsy was significantly lower (p=0.05) in treated-infected animals than in controls. In both protocols, the number of CD4+ T cells was lower at viremia peak in infected than in protected animals.

Expression of HIV-1 envelope gene by recombinant avipox viruses

Recombinant canarypox (CP) and fowlpox (FP) viruses that contained two forms of the HIV-1 (SF2 strain) env gene were engineered and their expression analysed in chick, simian and human cells. These vectors can efficiently replicate in avian but not in mammalian cells, in which infection is abortive. The two forms, consisting of the entire env open reading frame (IS+) or of the same gene lacking the putative immunosuppressive (IS-) region (amino acids 583-599), were individually inserted into the two virus vector backgrounds. In order to avoid premature transcription termination of the foreign gene and to improve protein expression, a mutagenesis was also performed within the T5NT motif without altering the amino acid sequence. By immunoprecipitation analyses, cells infected with CP and FP recombinants expressed HIV-1 env polypeptides of the appropriate molecular weight. We observed that the gp160 precursor was proteolytically cleaved except in MRC-5 cells infected with the IS- recombinants and that these polypeptides were glycosylated. Further analysis of these recombinant viruses by indirect immunofluorescence and syncytia inhibition assays indicated that the gp120 gp41 complex was present on the surface of infected cells, the number of syncytia being significantly lower when cells were infected by the CPIS- or FPIS- recombinants. Moreover, sera of immunized rabbits revealed the presence of specific antibodies in animals inoculated either with CP or with FP recombinants. These new constructs, which are unable to support a productive infection in human cells, might therefore also be a good anti-HIV-1 candidate vaccine in seropositive hosts.

Mycological and ultrastructural studies to evaluate biodeterioration of mural paintings. Detection of fungi and mites in Frescos of the monastery of St Damian in Assisi

Abstract The role of fungi in the biodeterioration of frescos has been investigated by microbiological and ultrastructural techniques. With this aim, the mycoflora present on samples taken from deteriorated indoor wall paintings (frescos) in the Monastery of St Damian in Assisi was isolated and identified. The results showed that the fungal colonization of the two paintings ‘Crocifisso con Francesco giovane’ and the ‘Mensa di S. Chiara’, located inside St Clares Refectory, was mainly due to Cladosporium, Aspergillus, Alternaria, Penicillium and Fusarium genera. Although considered not uncommon in frescos, their direct involvement in enzymatic degradation of paints was not confirmed. Interestingly, the presence of mites in proximity to actively growing fungal mycelium, as revealed by scanning electron microscopy, although considered an example of parasitic nutritional relation, could also suggest a metabolic interaction resulting in the synthesis of biodeteriogenic substances.

Canarypox and fowlpox viruses as recombinant vaccine vectors: A biological and immunological comparison

Canarypox and fowlpox viruses represent alternative vaccine vectors due to their natural host-range restriction to avian species. Although they cannot replicate in mammals, they correctly express transgenes in human cells and elicit a complete immune response in vaccinated subjects. Several studies have evaluated their genomic differences and protective efficacy in preclinical trials, but detailed information is not available for their transgene expression, cytokine modulation and abortive replication in mammals. This study demonstrates that the heterologous HIV gag/pol and env genes are more efficiently expressed by fowlpox in non-immune and immune cells. The production of retrovirus-like particles, the longer transgene expression, and a balanced cytokine induction may confer to fowlpox-based recombinants the ability to elicit a better immune response.

Transgenic chloroplasts are efficient sites for high‐yield production of the vaccinia virus envelope protein A27L in plant cells†

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.

Comparative analysis of immune responses and cytokine profiles elicited in rabbits by the combined use of recombinant fowlpox viruses, plasmids and virus-like particles in prime-boost vaccination protocols against SHIV.

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.

Prior DNA immunization enhances immune response to dominant and subdominant viral epitopes induced by a fowlpox-based SIVmac vaccine in long-term slow-progressor macaques infected with SIVmac251

A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy (ART) should be able to replenish virus-specific CD4+ T-cells and broaden the virus-specific CD8+ T-cell response in order to maintain CD8+ 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 CD4+ 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-(CD4+) T-cells able to produce TNF-alpha. Hopefully, this modality of vaccine combination may be useful in the clinical management of HIV-1-infected individuals.

THE ROLE OF TYPE-1 AND TYPE-2 T-HELPER IMMUNE RESPONSES IN HIV-1 VACCINE PROTECTION

Abstract: The dichotomy of type‐1 and type‐2 T‐helper (Th) immune responses is thought to be an obstacle to develop Human immunodeficiency virus‐type‐1 (HIV‐1) vaccines capable of inducing effective cellular as well as humoral immune responses. Macaca mulatta were immunized using two different HIV‐1sf2 envelope vaccine strategies, based on either immune‐stimulating complexes (ISCOM) or chimeric Fowlpox (FP) vaccines. One month following the third immunization all animals were heterologously challenged with simian/human immunodeficiency virus (SHIVsf13). Vaccinated monkeys, which were protected had the highest levels of both type‐1 and type‐2 HIV‐1 specific T‐helper cell (Th) responses in addition to the highest homologous and heterogenous virus neutralizing antibodies. To determine how long Th responses persisted and if they correlated with protection, animals were re‐challenged after waiting for four months without re‐boosting. Macaques which maintained the highest gp120‐specific type‐1 (IFN‐γ) responses were protected, while there was evidence of viral clearance in two others. These findings demonstrate the importance of both or mixed type‐1 and type‐2 Th responses in HIV‐1 vaccine induced immunity while suggesting a possible role of persistent type‐1 responses in maintaining protective immunity over time.

Construction and characterization of recombinant fowlpox viruses expressing human papilloma virus E6 and E7 oncoproteins.

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.