A lentiviral vector-based, herpes simplex virus 1 (HSV-1) glycoprotein B vaccine affords cross-protection against HSV-1 and HSV-2 genital infections.

Genital herpes is caused by herpes simplex virus 1 (HSV-1) and HSV-2, and its incidence is constantly increasing in the human population. Regardless of the clinical manifestation, HSV-1 and HSV-2 infections are highly transmissible to sexual partners and enhance susceptibility to other sexually transmitted infections. An effective vaccine is not yet available. Here, HSV-1 glycoprotein B (gB1) was delivered by a feline immunodeficiency virus (FIV) vector and tested against HSV-1 and HSV-2 vaginal challenges in C57BL/6 mice. The gB1 vaccine elicited cross-neutralizing antibodies and cell-mediated responses that protected 100 and 75% animals from HSV-1- and HSV-2-associated severe disease, respectively. Two of the eight fully protected vaccinees underwent subclinical HSV-2 infection, as demonstrated by deep immunosuppression and other analyses. Finally, vaccination prevented death in 83% of the animals challenged with a HSV-2 dose that killed 78 and 100% naive and mock-vaccinated controls, respectively. Since this FIV vector can accommodate two or more HSV immunogens, this vaccine has ample potential for improvement and may become a candidate for the development of a truly effective vaccine against genital herpes.

Role of hematopoietic cells in the maintenance of chronic human torquetenovirus plasma viremia.

Many aspects of the life cycle of torquetenoviruses (TTVs) are essentially unexplored. In particular, it is still a matter of speculation which cell type(s) replicates the viruses and maintains the generally high viral loads found in the blood of infected hosts. In this study, we sequentially measured the TTV loads in the plasma of four TTV-positive leukemia patients who were strongly myelosuppressed and then transplanted with haploidentical hematopoietic stem cells. The findings provide clear quantitative evidence for an extremely important role of hematopoietic cells in the maintenance of TTV viremia.

Env-expressing autologous T lymphocytes induce neutralizing antibody and afford marked protection against feline immunodeficiency virus.

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 x 10(6) to 10 x 10(6) viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.

Human anelloviruses and the central nervous system.

Torque teno virus and related anelloviruses are a recent addition to the list of agents that cause chronic productive infections and high levels of plasma viraemia in humans. Many aspects of the natural history and pathogenesis of these under many respects surprising viruses are still poorly understood. In this review, we briefly outline the general properties of anelloviruses, examine what is currently known about the interactions they establish with the central nervous system (CNS), and discuss the possible pathological consequences.

A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts.

BACKGROUND: Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major histocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry. RESULTS: The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies. CONCLUSION: Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

A dormant internal ribosome entry site controls translation of feline immunodeficiency virus.

The characterization of internal ribosome entry sites (IRESs) in virtually all lentiviruses prompted us to investigate the mechanism used by the feline immunodeficiency virus (FIV) to produce viral proteins. Various in vitro translation assays with mono- and bicistronic constructs revealed that translation of the FIV genomic RNA occurred both by a cap-dependent mechanism and by weak internal entry of the ribosomes. This weak IRES activity was confirmed in feline cells expressing bicistronic RNAs containing the FIV 5' untranslated region (UTR). Surprisingly, infection of feline cells with FIV, but not human immunodeficiency virus type 1, resulted in a great increase in FIV translation. Moreover, a change in the cellular physiological condition provoked by heat stress resulted in the specific stimulation of expression driven by the FIV 5' UTR while cap-dependent initiation was severely repressed. These results reveal the presence of a "dormant" IRES that becomes activated by viral infection and cellular stress.

Immunotherapy with internally inactivated virus loaded dendritic cells boosts cellular immunity but does not affect feline immunodeficiency virus infection course.

Immunotherapy of feline immunodeficiency virus (FIV)-infected cats with monocyte-derived dendritic cells (MDCs) loaded with aldrithiol-2 (AT2)-inactivated homologous FIV was performed. Although FIV-specific lymphoproliferative responses were markedly increased, viral loads and CD4+ T cell depletion were unaffected, thus indicating that boosting antiviral cell-mediated immunity may not suffice to modify infection course appreciably.

Torquetenovirus infection and ciliary dysmotility in children with recurrent pneumonia.

BACKGROUND: The aim of the study was to assess Torquetenovirus (TTV) loads within respiratory ciliated cells and to verify the existence of a correlation between TTV loads and functional or structural ciliary abnormalities, in a group of children with recurrent or persistent pneumonia. METHODS: Nasal brushing samples of 55 children (28 male) were evaluated for ciliary motion and ultrastructural assessment, as well as for detection and quantification of TTV. Moreover, presence and load of TTV within ciliated cells, obtained from 5 patients by laser capture microdissection, were determined. RESULTS: The nasal samples of 47 (85%) children with persistent or recurrent pneumonia resulted positive for TTV (loads = 2.1-7.3 log10 copies/microg total DNA). TTV were demonstrated also within microdissected ciliated cells. No significant difference between primary (11 subjects) and secondary ciliary dyskinesia (44 subjects) for TTV prevalence and mean loads were found. A significant correlation was observed between nasal TTV loads and ciliary beat frequency score (r = 0.305; P < 0.05), but not between TTV loads and presence of abnormal motion patterns, in patients with secondary ciliary abnormalities. As expected no correlations were found between nasal TTV loads and ciliary motion analysis in primary ciliary dyskinesia. CONCLUSIONS: The presence of TTV in nasal samples demonstrates TTV ability to infect respiratory ciliated cells and suggests that these cells are potentially able to support virus replication. Moreover, TTV may behave in respiratory cells in a similar way to other viruses, that is, they disrupt the mucociliary escalator.

Physicochemical characterization of a peptide deriving from the glycoprotein gp36 of the feline immunodeficiency virus and its lipoylated analogue in micellar systems.

P59 is the Trp-rich 20-mer peptide ((767)L-G(786)), partial sequence of the membrane-proximal external region (MPER) of the FIV gp36. It has potent antiviral activity, possibly due to a mechanism that inhibits the fusion of the virus with the cell membranes. In the hypothesis that a lipophilic tail could enhance the adhesion of P59 to the membrane so improving its antiviral activity, we synthesized its lipoylated analogue lipo-P59. Fluorescence, CD and NMR investigations in membrane mimicking environments (such as SDS and DPC micelles) were aimed to assess the potential of the lipo-P59 lipophilic tail to affect the biophysical and conformational behaviour of the peptide. In vitro inhibitory assays using lymphoid cell cultures to check the antiviral activity of peptides were also performed. The data show that the biophysical properties and the conformational preferences of the peptides are not dramatically affected by the hydrophobic tail, suggesting that the lipopeptide is capable of preserving all the biophysical peculiarities. Similarly, antiviral experimental data show that the membrane-anchored lipo-P59 peptide is also effective in inhibiting virus replication. Moreover, the lipophilic tail allows P59 to preserve its antiviral activity even in conditions in which the non lipoylated peptide is devoid of activity. In accordance with the unusual high Trp presence, the peptides confirm the preference to be positioned on the membrane interface. Furthermore, the data point out a peculiarity of interaction of the peptides with SDS as compared with DPC.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS.

BACKGROUND: Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. METHODS: Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. RESULTS: The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. CONCLUSION: We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in human clinical trials, as one of the most potent anti-FIV agents ever tested in vitro. This finding may provide new avenues for treating FIV infection and contribute to the development of a small animal model mimicking the effects of ART in humans.

Human bocavirus in Italian patients with respiratory diseases.

BACKGROUND: hBoV, a recently discovered parvovirus, can be present in the respiratory tract of patients with acute respiratory diseases (ARD), but its etiologic involvement in the underlying diseases is still uncertain. OBJECTIVE: To determine in a retrospective study, the prevalence of hBoV, compared with common respiratory viruses (RV), in respiratory specimens from patients with ARD. STUDY DESIGN: A total of 335 specimens obtained over 7 years were examined. Two hundred were nasal swabs from infants hospitalized for ARD, 84 were nasal swabs or bronchoalveolar lavages from adults with pneumonia, bronchopneumonia or asthma, and 51 were nasal swabs from healthy children. RESULTS: The overall rate of hBoV detection in specimens from infants with ARD, which was 4.5%, varied slightly from year to year, except for the period 2000-2002, when no specimen was positive. Unlike other RV, no seasonal variation in hBoV incidence was noted. Infants with hBoV infection suffered either from bronchiolitis or from bronchopneumonia and 5 out of 9 cases yielded no co-infecting viral pathogen. Only one sample from an adult was hBoV positive. None of the nasal swabs from healthy subjects tested hBoV-positive. CONCLUSIONS: The findings indicate that hBoV can cause ARD in infants.

Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes.

BACKGROUND: Safe and efficient vector systems for delivering antigens or immunomodulatory molecules to dendritic cells (DCs), T lymphocytes or both are considered effective means of eliciting adaptive immune responses and modulating their type, extent, and duration. As a possible tool toward this end, we have developed a self-inactivating vector derived from feline immunodeficiency virus (FIV) showing performance characteristics similar to human immunodeficiency virus-derived vectors but devoid of the safety concerns these vectors have raised. METHODS: The pseudotyped FIV particles were generated with a three-plasmid system consisting of: the packaging construct, providing Gag, Pol and the accessory proteins; the vector(s), basically containing FIV packaging signal (psi), Rev responsive element, R-U5 region at both ends, and the green fluorescent protein as reporter gene; and the Env plasmid, encoding the G protein of vesicular stomatitis virus (VSV-G) or the chimeric RD114 protein. Both packaging and vector constructs were derived from p34TF10, a replication competent molecular clone of FIV. The pseudotyped particles were produced by transient transfection in the Crandell feline fibroblast kidney (CrFK) or the human epithelial (293T) cell line. RESULTS: To broaden its species tropism, the final vector construct was achieved through a series of intermediate constructs bearing a longer psi, the FIV central polypurin tract sequence (cPPT), or the woodchuck hepatitis post-regulatory element (WPRE). These constructs were compared for efficiency and duration of transduction in CrFK or 293T cells and in the murine fibroblast cell line NIH-3T3. Whereas psi elongation and cPPT addition did not bring any obvious benefit, insertion of WPRE downstream GFP greatly improved vector performances. To maximize the efficiency of transduction for ex-vivo murine DCs and T-lymphocytes, this construct was tested with VSV-G or RD114 and using different transduction protocols. The results indicated that the FIV construct derived herein stably transduced both cell types, provided that appropriate vector makeup and transduction protocol were used. Further, transduced DCs underwent changes suggestive of an induced maturation. CONCLUSION: In contrast to previously described FIV vectors that were poorly efficient in delivering genetic material to DCs and T lymphocytes, the vector developed herein has potential for use in experimental immunization strategies.

High torquetenovirus loads are correlated with bronchiectasis and peripheral airflow limitation in children.

BACKGROUND: The aim of the study was to evaluate the prevalence of torquetenovirus (TTV) infection in a group of children with recurrent lower respiratory tract infections and radiologic evidence of bronchiectasis. Correlations between TTV loads and severity of bronchiectasis and between TTV loads and lung function were evaluated. METHODS: In 38 subjects, high-resolution computed tomography (HRCT) and plasma tests for TTV detection and quantification were done. In 21/38 subjects, spirometry was also performed. RESULTS: TTV was found in 31/38 (81.6%) patients. The correlation between TTV loads and severity of bronchiectasis was statistically significant (r = 0.548; P = 0.01). TTV loads showed inverse correlation with FEF25-75% (r = -0.541; P = 0.011), and FEF25-75%/FVC (r = -0.512; P = 0.018). Inverse correlation was found also between severity of bronchiectasis and functional lung parameters: FEF25-75% (r = -0.635; P = 0.002), FEV1/FVC (r = -0.541; P = 0.011), and FEF25-75%/FVC (r = -0.645; P = 0.002). CONCLUSIONS: This study demonstrated the high prevalence of TTV infection in children with bronchiectasis. Moreover, we have shown a significant correlation between TTV loads and airflow limitation within the peripheral airways, as well as between severity of bronchiectasis and decrease of lung function.

Feline immunodeficiency virus plasma load reduction by a retroinverso octapeptide reproducing the Trp-rich motif of the transmembrane glycoprotein.

The Trp-rich motif (TrpM) of the transmembrane glycoprotein (TM) of lentiviruses is an attractive domain on which to design new potential cell entry peptide inhibitors. We recently demonstrated that an octapeptide reproducing the TrpM of feline immunodeficiency virus (FIV), designated C8, broadly inhibited this virus in vitro and that the retroinverso analogue of this peptide (riC8) was almost as inhibitory and exhibited features suggestive of a much increased stability. Here, we demonstrated that riC8 is indeed highly stable, maintaining its concentration unchanged for at least 24 h in cat serum in vitro. Furthermore, once inoculated into cats, riC8 produced no major acute toxic effects and exhibited satisfactory pharmacokinetic properties. Finally, we report the results of a short-term monotherapy experiment in chronically FIV-infected cats showing that riC8 is well tolerated and also has substantial antiviral activity in vivo. In particular, the mean viral load of riC8-treated animals declined progressively with increasing time of treatment, whereas that of control animals given C8 or solvent alone did not. These results provide the first evidence that clinically useful inhibition of virus replication with a small peptide derived from a functional domain of the TM of a lentivirus can be achieved in vivo.

Retroinverso analogue of the antiviral octapeptide C8 inhibits feline immunodeficiency virus in serum.

We described the antiviral activity of an octapeptide corresponding to a Trp-rich domain of feline immunodeficiency virus (FIV) transmembrane glycoprotein. To overcome the limited enzymatic stability of short peptides, the retroinverso analogue was prepared and tested for inhibitory activity of FIV in the presence or absence of normal cat serum. Differently from the unmodified peptide, the retroinverso analogue maintains strong inhibitory activity in serum. NMR studies showed that it displays crucial conformational features believed to be important for antiviral activity.

Genetic and phylogenetic analyses of HIV-1 corroborate the transmission link hypothesis.

BACKGROUND: Phylogenetic and genetic analyses have proven a valuable tool to infer epidemiological links between human immunodeficiency virus type-1 (HIV-1) isolates. These methods were applied in the present report for studying the genetic relatedness of the viral strains involved in two episodes of suspected HIV-1 transmission. OBJECTIVES: Provide any evidence that may help establish or refute the transmission link. STUDY DESIGN: In the first case, a leukemic patient became HIV-1 positive following the transfusion of platelets from a donor who was subsequently found to have tested false HIV-seronegative and to be sexual partner to an infected woman. In the second, a wife claimed to have acquired the infection from her husband who had concealed his infected status. RESULTS AND CONCLUSIONS: The viral pairs detected in each of the suspected transmission cases exhibited common amino acid signatures and low genetic distances and segregated together in phylogenetic trees, thus showing a level of genetic relatedness similar to reference pairs known with certainty to be epidemiologically linked. These findings corroborated the existence of a direct transmission link in both the episodes with a high level of confidence.

Torque teno virus viremia load size in patients with selected congenital defects of innate immunity.

Plasma loads of torque teno virus (TTV) among individuals differ extensively beginning early in life, suggesting a role for innate immunity. Here, congenital mannose-binding lectin deficiencies, but not deficiencies in respiratory ciliary function, correlated with increased TTV loads. Notably, however, the presence of either disorder was associated with particularly high TTV loads.