Eradication of pre-established lymphoma using herpes simplex virus amplicon vectors

Herpes simplex virus amplicon vectors expressing RANTES (HSVrantes) and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for their ability to elicit a tumor-specific T-cell response in a murine lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed high levels of B7.1 and RANTES as analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells inoculated contralaterally. Direct intratumoral injection of HSVB7.1 and/or HSVrantes alone or in combination into established EL4 tumors led to complete tumor regression in injected tumors as well as in nontransduced contralaterally implanted tumor, whereas control tumors or tumors injected with HSVlac expressing beta-galactosidase did not regress. Maximal protection was achieved with combined injection of HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to rechallenge with parental EL4 cells, and tumor cell-specific cytolytic T-cell activity was observed in mice demonstrating regression. HSV amplicon-mediated delivery of immune effector molecules may represent a useful strategy for immunotherapy in the setting of pre-existing tumor.     

Inducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system

Herpes simplex virus-based amplicon vectors have been used for gene transfer into cultured neurons and the adult CNS. Since constitutive expression of a foreign gene or overexpression of an endogenous gene may have deleterious effects, the ability to control temporal expression would be advantageous. To achieve inducible gene expression, we have incorporated the tetracycline-responsive promoter system into amplicon vectors and showed, both in vitro and in vivo, that expression can be modulated by tetracycline. Using the firefly luciferase as the reporter gene, maximal repression by tetracycline in hippocampal cultures was about 50-fold. Withdrawal of tetracycline derepressed gene expression, reaching maximal levels within 10-12 h. In contrast, addition of tetracycline to cultures without prior tetracycline exposure inhibited gene expression rapidly; luciferase activity was reduced to less than 8% within 24 h. In adult rat hippocampus, vectors expressing luciferase or the Escherichia coli lacZ were repressed by tetracycline 9- and 60-fold, respectively. Maximum gene expression from the vectors occurred 2-3 days post-infection and declined thereafter. Such decline impeded further induction of expression by withdrawing tetracycline. This study demonstrates the feasibility of incorporating a powerful inducible promoter system into HSV vectors. The development of such an inducible viral vector system for gene transfer into the adult CNS might prove to be of experimental and therapeutic value.     

ATP depletion blocks herpes simplex virus DNA packaging and capsid maturation

During herpes simplex virus (HSV) assembly, immature procapsids must expel their internal scaffold proteins, transform their outer shell to form mature polyhedrons, and become packaged with the viral double-stranded (ds) DNA genome. A large number of virally encoded proteins are required for successful completion of these events, but their molecular roles are poorly understood. By analogy with the dsDNA bacteriophage we reasoned that HSV DNA packaging might be an ATP-requiring process and tested this hypothesis by adding an ATP depletion cocktail to cells accumulating unpackaged procapsids due to the presence of a temperature-sensitive lesion in the HSV maturational protease UL26. Following return to permissive temperature, HSV capsids were found to be unable to package DNA, suggesting that this process is indeed ATP dependent. Surprisingly, however, the display of epitopes indicative of capsid maturation was also inhibited. We conclude that either formation of these epitopes directly requires ATP or capsid maturation is normally arrested by a proofreading mechanism until DNA packaging has been successfully completed.     

Genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D

Intramuscular injection of mice with an adeno-associated virus (AAV) vector expressing herpes simplex virus type 2 glycoprotein B led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses.     

DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508-519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.     

Gastritis secondary to herpes simplex virus

Gastric infection with herpes simplex virus is rare, with only two cases previously reported. At the time of the previous reports, the virus could not be cultured, and the diagnosis was based on histological findings. Two cases of culture positive herpes simplex virus gastritis are presented, emphasizing the importance of routine gastric biopsies and viral cultures in immunodeficient patients with dyspeptic symptoms.     

Physical and functional interactions between the herpes simplex virus UL15 and UL28 DNA cleavage and packaging proteins

Herpes simplex virus (HSV) DNA is cleaved from concatemers and packaged into capsids in infected cell nuclei. This process requires seven viral proteins, including UL15 and UL28. UL15 expressed alone displays a nuclear localization, while UL28 remains cytoplasmic. Coexpression with UL15 enables UL28 to enter nuclei, suggesting an interaction between the two proteins. Additionally, UL28 copurified with UL15 from HSV-infected cells after ion-exchange and DNA affinity chromatography, and the complex sedimented as a 1:1 heterodimer upon sucrose gradient centrifugation. These findings are evidence of a physical interaction of UL15 and UL28 and a functional role for UL15 in directing UL28 to the nucleus.     

A packaging system for SV40 vectors without viral coding sequences

We studied the incidence of regurgitation in 100 patients undergoing elective gynecological laparoscopies under general anesthesia with intermittent positive pressure ventilation using a laryngeal mask airway (LMA). Patients ingested methylene blue capsules 10-15 min before induction of anesthesia. After induction and insertion of an LMA using the recommended insertion technique, a fiberoptic examination of the larynx was made for traces of dye and to site a pH probe in the bowl of the LMA for continuous monitoring. LMA insertion was successful in all patients within two attempts (95 at first attempt). Fiberoptic examination revealed the vocal cords or cords and posterior or anterior epiglottis in 96 and no trace of dye in 99 patients. One patient regurgitated dye immediately after induction, and the stain was seen on the LMA after removal. The remaining 99 LMAs were not stained. Thirty patients were randomly selected for fiberoptic examination of the laryngopharynx before neuromuscular block was antagonized. Methylene blue staining did not occur in any of these patients. In 91 patients with complete pH data, regurgitation (pH < 4.0) did not occur. The 95% confidence limit for a true probability of regurgitation in this study is 0.041 or a true rate of regurgitation of less than 4.1%. A larger study would be required to possibly demonstrate a lower incidence of regurgitation. This study confirms the clinical impression that the incidence of regurgitation during laparoscopies with a LMA is extremely low.     

GABA synthesis in astrocytes after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase 65 or 67

Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform-specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a beta-galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. GABA was detected in glial fibrillary acid protein-expressing cells after GAD65 vector infection. Significant amounts of GABA, as determined by HPLC, were synthesized in cultures infected with either GAD vector. The levels of GABA in GAD67 vector-infected cells were almost twofold higher than in GAD65 vector-infected cells. Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K+, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.     

Analysis of a multi-mutant herpes simplex virus type 1 for gene transfer into sympathetic preganglionic neurons and a comparison to adenovirus vectors

A non-replicating triple-mutant herpes simplex virus (14H delta 3vhsZ) expressing the bacterial marker enzyme beta-galactosidase, was assessed for neurotropism and cytopathic effects as a vector for gene transfer into differentiated phaeochromocytoma 12 cells in vitro and into spinal sympathetic neurons in vivo. In the in vivo study, the 14H delta 3vhsZ was injected into the adrenal gland of hamsters. For comparison, an evaluation of two adenovirus vectors, AdCA17lacZ and AdCA36lacZ, was performed. Infection of the differentiated phaeochromocytoma 12 cells by 14H delta 3vhsZ resulted in intense beta-galactosidase staining in 80-90% of the cells without changes in cell morphology, detected by light microscopy, after a period of four days. No cytoskeletal disruption was detected by immunocytochemistry for the neurofilament protein and no apoptosis was demonstrated by the Hoescht stain for nuclear chromatin in virus-infected cells in comparison to mock-infected control cells. Twoto three days after adrenal inoculation with 14H delta 3vhsZ, beta-galactosidase was detected in 240 preganglionic neurons per hamster (n = 8), a number equal to about 25% of the population of targeted neurons. The beta-galactosidase reaction product extended throughout the normal kite-shaped neuronal somata and extensive dendritic arbour. The number decreased to 120 by five days (n = 3) and to two by eight days (n = 4). This decrease was presumably due to loss of expression of the marker gene and not to cell death because, at eight days, the number of sympathetic pregnanglionic neurons in the nucleus intermediolateralis, pars principalis, that were immunoreactive for the neurotransmitter enzyme choline acetyltransferase, and demonstrated nicotinamide adenine dinucleotide phosphate-diaphorase activity, were the same on the infected left side of the cord as on the uninfected right side. Inflammatory cells surrounded some of the infected neurons at five days but by eight days the infiltrate was reduced. Infection of differentiated phaeochromocytoma 12 cells by AdCA17lacZ and AdCA36lacZ also resulted in marker gene expression in a large proportion of the cells (80-90%) in the absence of cytopathic effects. In contrast, four days after adrenal injection of AdCA17lacZ or AdCA36lacZ (n = 5 for each) only an average of three preganglionic neurons per hamster expressed beta-galactosidase activity, despite clear adrenal infection. AdCA17lacZ and AdCA36lacZ both produced light patches of staining confined to the neuronal soma. These neurons had normal morphology but sometimes were surrounded by an inflammatory infiltrate. In conclusion, the non-replicating herpes simplex virus, 14H delta 3vhsZ, had minimal cytotoxic effects in neurons, in vitro or in vivo, and was efficiently transported from the adrenal gland to infect many sympathoadrenal pregnanglionic neurons. In contrast, very few neurons demonstrated beta-galactosidase activity after injection into the adrenal gland of AdCA17lacZ and AdCA36lacZ. Therefore, 14H delta 3vhsZ is a more suitable vector than either of the adenovirus vectors tested for eliciting short-term changes in preganglionic neuron gene expression.     

DNA immunization against experimental genital herpes simplex virus infection

A nucleic acid vaccine, expressing the gene encoding herpes simplex virus (HSV) type 2 glycoprotein D (gD2) under control of the cytomegalovirus immediate-early gene promoter, was used to immunize guinea pigs against genital HSV-2 infection. The vaccine elicited humoral immune responses comparable to those seen after HSV-2 infection. Immunized animals exhibited protection from primary genital HSV-2 disease with little or no development of vesicular skin lesions and significantly reduced HSV-2 replication in the genital tract. After recovery from primary infection, immunized guinea pigs experienced significantly fewer recurrences and had significantly less HSV-2 genomic DNA detected in the sacral dorsal root ganglia compared with control animals. Thus, immunization reduced the burden of latent infection resulting from intravaginal HSV-2 challenge, and a nucleic acid vaccine expressing the HSV-2 gD2 antigen protected guinea pigs against genital herpes, limiting primary infection and reducing the magnitude of latent infection and the frequency of recurrent disease.     

Inhibition of herpes simplex virus replication by retinoic acid

Superior laryngeal nerve (SLN) stimulation can activate the brainstem swallowing mechanism to produce a complete swallowing sequence consisting of oropharyngeal, oesophageal and lower oesophageal sphincter (LOS) components. However, little is known of the effect of SLN stimulation (peripheral-sensory input from the pharynx) on the characteristics of oesophageal motor activity, especially in the smooth muscle portion. The present study examined the effect of varying stimulus train length and frequency on each of the three components of the reflex. Acute studies were performed in urethane anaesthetized cats. Oesophageal motility was monitored using conventional manometric techniques, and oropharyngeal swallowing by the mylohyoid electromyogram. SLN stimulus train length (1-10 sec) and frequency (5-30 Hz) were varied independently. Increased train length or frequency resulted in (1) an increase in oropharyngeal swallowing and incidence of the complete swallowing response, (2) an increase in latency to onset of the oesophageal peristaltic wave, (3) reduction of the amplitude of the evoked peristaltic contraction in the smooth muscle portion, without altering its velocity, (4) increased LOS relaxation, and increased LOS after-contraction. The LOS contraction was abolished by atropine (100 micrograms kg-1). Therefore, increased SLN stimulation not only results in excitation of the central swallowing program and the oropharyngeal stage of swallowing, but has major effects on the oesophageal and LOS stages of swallowing. Afferent SLN stimuli can impact on the control mechanisms for each stage, to inhibit or excite the stages in different ways.     

Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.     

Functional analysis of the herpes simplex virus UL42 protein

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

Cellular protein interactions with herpes simplex virus type 1 oriS

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.