Immunogenic activity of temperature sensitive mutation of herpes simplex virus type-1 in mono- and polyvalent conditions with different attenuated strains of virus. II. Analysis of humoral immunologic response induced by mutants of temperature sensitive herpes simplex type-1 virus in polyvalent systems

The ability of mutant ts HSV-1 on 45 passage to induction of IgG antibody response in the presence of other attenuated viral strain, like measles, mumps, rubella was evaluated, and the effect of mutant ts HSV-1 on humoral immunity induced by attenuated measles and mumps strains was also tested. The obtained results clearly indicated that even immunization with other RNA virus strains had no effect on IgG antibody response to mutant ts HSV-1. Significant differences were observed in IgG antibody responses to mumps and measles between groups of guinea pigs immunized with these viruses alone or together with other viruses. This was especially observed in the case of mumps virus. It clearly shows that change of virus concentration rates in polyvalent schema of immunization is needed.     

Immunogenic activity of temperature sensitive mutation of herpes simplex virus type-1 in mono- and polyvalent conditions with different attenuated strains of virus. I. Analysis of humoral immunity induced by mutant temperature sensitive herpes simplex virus type-1 in monovalent systems

The aim of the present study was the analysis of the immunogenic activity of temperature sensitive mutant of HSV-1 with significantly decreased pathogenicity for mice. The stability of immunogenic potency was also tested. The level of antibody-mediated response in guinea pigs after two doses of mutant ts HSV-1 or native strain was compared. Antibodies to HSV were measured using ELISA and CFM. Seroconversion rates in tested groups were high, from 80% to 100%. The level of antibody response at 4 week after inoculation measured by ELISA and CFM showed no significant differences between 4 passages of mutant ts HSV-1. Significant higher increase of antibodies level after two doses of viruses was observed for mutant ts HSV-1 as compared to native strain, both in ELISA and CFM.     

Physical mapping of the herpes simplex virus type 2 nuc- lesion affecting alkaline exonuclease activity by using herpes simplex virus type 1 deletion clones

The nuc- lesion affecting alkaline exonuclease activity in the herpes simplex virus type 2 (HSV-2) mutant ts1348 had previously been mapped to the EcoRI-D restriction enzyme fragment of HSV-1. Eight clones with deletions representing most of HSV-1 EcoRI fragment D were selected with lambda gtWES hybrids. These clones were tested for their ability to rescue the alkaline exonuclease activity of HSV-2 nuc- ts1348 virus. The sequences colinear with the HSV-2 nuc- lesion were found to map between 0.169 and 0.174 map units on the HSV-1 Patton genome, representing an 0.8-kilobase-pair region that is 12.9 to 13.7 kilobase pairs from the left end of HSV-1 EcoRI fragment D.     

Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection

Immunization of mice with herpes simplex virus type 1 (HSV-1) mutant viruses containing deletions in the gene for virion host shutoff (vhs) protein diminishes primary and recurrent corneal infection with wild-type HSV-1. vhs mutant viruses are severely attenuated in vivo but establish latent infections in sensory neurons. A safer HSV-1 mutant vaccine strain, Delta41Delta29, has combined vhs and replication (ICP8-) deficits and protects BALB/c mice against primary corneal infection equivalent to a vhs- strain (BGS41). Here, we tested the hypothesis that Delta41Delta29 can protect as well as BGS41 in a therapeutic setting. Because immune response induction varies with the mouse and virus strains studied, we first determined the effect of prophylactic Delta41Delta29 vaccination on primary ocular infection of NIH inbred mice with HSV-1 McKrae, a model system used to evaluate therapeutic vaccines. In a dose-dependent fashion, prophylactic Delta41Delta29 vaccination decreased postchallenge tear film virus titers and ocular disease incidence and severity while eliciting high levels of HSV-specific antibodies. Adoptive transfer studies demonstrated a dominant role for immune serum and a lesser role for immune cells in mediating prophylactic protection. Therapeutically, vaccination with Delta41Delta29 effectively reduced the incidence of UV-B-induced recurrent virus shedding in latently infected mice. Therapeutic Delta41Delta29 and BGS41 vaccination decreased corneal opacity and delayed-type hypersensitivity responses while elevating antibody titers, compared to controls. These data indicate that replication is not a prerequisite for generation of therapeutic immunity by live HSV mutant virus vaccines and raise the possibility that genetically tailored replication-defective viruses may make effective and safe therapeutic vaccines.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.     

Herpes simplex virus DNA polymerase as the site of phosphonoacetate sensitivity: temperature-sensitive mutants.

Temperature-sensitive (ts) mutants in a number of complementation groups of herpes simplex virus type 1 (HSV-1) are deficient in DNA polymerase induction at the restrictive temperature. Twenty-two mutants in 15 complementation groups were tested for sensitivity to phosphonoacetate (PAA), a compound that inhibits HSV replication in vivo and the DNA polymerase in vitro. One mutant, tsD9, was resistant to PAA (Pr), whereas all others were sensitive. Revertants of tsD9 to the ts+ phenotype simultaneously lost PAA resistance. Additional Pr mutants were isolated from ts mutants belonging to several complementation groups of HSV-1. Double mutants (ts Pr phenotype) were used in three-factor recombination analyses to locate the PAA locus on the genetic map at a position indistinguishable from the ts lesion in tsD9. In all cases, resistance or sensitivity to PAA in vivo was correlated with resistance or sensitivity of DNA polymerase in vitro. These data are compatible with the temperature-sensitive lesion of tsD9 and the determinant of PAA sensitivity both residing in the structural gene for DNA polymerase.     

Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome.

Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature.     

Selective ablation of virion host shutoff protein RNase activity attenuates herpes simplex virus 2 in mice

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.     

Interaction of herpes simplex virus type 2 with a rat glioma cell line

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.     

Immunization with replication-defective mutants of herpes simplex virus type 1: sites of immune intervention in pathogenesis of challenge virus infection.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) were used as a new means to immunize mice against HSV-1-mediated ocular infection and disease. The effects of the induced immune responses on pathogenesis of acute and latent infection by challenge virus were investigated after corneal inoculation of immunized mice with virulent HSV-1. A single subcutaneous injection of replication-defective mutant virus protected mice against development of encephalitis and keratitis. Replication of the challenge virus at the initial site of infection was lower in mice immunized with attenuated, wild-type parental virus (KOS1.1) or replication-defective mutant virus than in mice immunized with uninfected cell extract or UV-inactivated wild-type virus. Significantly, latent infection in the trigeminal ganglia was reduced in mice given one immunization with replication-defective mutant virus and was completely prevented by two immunizations. Acute replication in the trigeminal ganglia was also prevented in mice immunized twice with wild-type or mutant virus. The level of protection against infection and disease generated by immunization with replication-defective mutant viruses was comparable to that of infectious wild-type virus in all cases. In addition, T-cell proliferative and neutralizing antibody responses following immunization and corneal challenge were of similar strength in mice immunized with replication-defective mutant viruses or with wild-type virus. Thus, protein expression by forms of HSV-1 capable of only partially completing the replication cycle can induce an immune response in mice that efficiently decreases primary replication of virulent challenge virus, interferes with acute and latent infection of the nervous system, and inhibits the development of both keratitis and systemic neurologic disease.     

Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line.

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody.     

Antigenic properties and cellular localization of herpes simplex virus glycoprotein H synthesized in a mammalian cell expression system.

Herpes simplex virus type 1 glycoprotein H (HSV-1 gH) was synthesized in an inducible mammalian cell expression system, and its properties were examined. The gH coding sequence, together with the stable 5' untranslated leader sequence from xenopus beta-globin, was placed under control of the strong promoter from the human cytomegalovirus major immediate-early gene in an amplifiable plasmid which contains the simian virus 40 (SV40) virus origin for replication (ori). This expression vector was transfected into ts COS cells constitutively expressing a temperature-sensitive SV40 T antigen which allows utilization of the SV40 ori at permissive temperatures. The results of transient expression assays at the permissive temperature showed that HSV-1 gH could be synthesized in greater amounts than those produced by a high-multiplicity virus infection. The proteins produced were detected in Western blots (immunoblots) with a HSV-1 gH-specific polyclonal serum raised against a TrpE-gH fusion protein. The transfected gH had an apparent molecular weight of approximately 105,000, intermediate in size to those of the precursor (100,000) and fully processed forms (110,000) of HSV-1 gH from infections. Antigenicity was investigated by reactions with three virus-neutralizing monoclonal antibodies specific for conformational epitopes on gH. Only one of these monoclonal antibodies could immunoprecipitate the synthesized gH. However, equal recognition of the transfected gH was achieved by superinfection with virus. In addition, detectable amounts of gH were not expressed on the cell surface unless the cells were superinfected with virus. Studies with a temperature-sensitive mutant, ts1201, defective in encapsidation showed that the changes in antigenic structure and cell surface expression caused by superinfection with virus were not due simply to incorporation of gH into virions. These results suggest that gH requires additional virus gene products for cell surface localization and formation of an antigenic structure important for its function in mediating infectivity.     

Alterations in the antigenic structure of two major HSV-1 glycoproteins, gC and gB, influence immune regulation and susceptibility to murine herpes keratitis

We previously demonstrated that anterior chamber (AC) injection of HSV-1 before or simultaneous with topical corneal HSV-1 infection resulted in cellular immune tolerance of HSV-1 Ag and a reduced frequency of corneal stromal lesions. In the present study, we have investigated the role of the HSV-1 cell-surface glycoproteins gC and gB in the induction of tolerance, and the resulting reduced susceptibility to HSV-1 corneal stromal disease. These studies utilized mutant strains of HSV-1 with deletion or point mutations in the gene coding for gC or gB. Groups of mice received topical corneal infections with wild-type HSV-1, followed by AC injection of the same eye with wild-type HSV-1 or a mutant strain. Varying the antigenic composition of the virus injected into the AC resulted in three distinct patterns of immune responsiveness. In agreement with our previous findings, AC injection of wild-type HSV-1 induced a state of HSV-1 specific tolerance that extended to both the delayed type hypersensitivity (DTH) and CTL responses. A mutant strain lacking gC (gC-) induced partial tolerance characterized by undetectable CTL activity but a normal DTH response. A mutant strain lacking gB (gB-) caused partial suppression of the CTL response and no reduction of the DTH response. Thus, whereas gB may be involved in CTL tolerance induction in this model, gC clearly is not involved. In contrast, both gC and gB must be present in the AC to induce detectable DTH tolerance. The latter interpretation was strengthened by the observation that AC injection of a mixture of gC- (expressing normal gB) and gB- (expressing normal gC) effectively suppressed the DTH response to wild-type HSV-1. A panel of mar mutants with individual point mutations affecting gC and gB was used to identify the epitopes responsible for induction of DTH tolerance. Two of the gC mutants failed to induce DTH tolerance to wild-type HSV-1 when injected into the AC, suggesting that the sites on the gC molecule that are altered by these mutations are important for the induction of DTH tolerance. Similarly, one of the mar mutants for gB uniformly failed to suppress the DTH response, while another had a variable effect. The unique pattern of cellular immune reactivity exhibited by the mice receiving simultaneous topical corneal infection with wild-type HSV-1 and AC injection of gC- (no CTL but normal DTH) was associated with significantly reduced susceptibility to HSV-1 corneal stromal lesions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2.

It is well-known that viral thymidine kinase (TK) expression is important for the maximum demonstration of virulence of herpes simplex virus (HSV). In this study, we have investigated interactions of a TK- mutant of virulent HSV type 2 (HSV-2) (syn+) and a nonneuroinvasive HSV-1 (syn) in mice. When the mice were inoculated with each virus alone in their rear footpads, no mice were killed even after infection with high doses of viruses (greater than 10(6) PFU per mouse), whereas 100% of the mice died when inoculated with 10(5) PFU of a 1:1 mixture of HSV-2 TK- mutant and nonneuroinvasive HSV-1. The 1:1 mixture exhibited even more virulence than the parental HSV-2; the mean survival time of coinfected mice was significantly shorter than that of mice inoculated with 10(5) PFU of the virulent HSV-2. We have also examined the genotypes and phenotypes of viruses isolated from the central nervous system of coinfected mice. Of 50 isolates, 7 were judged to be recombinants from their restriction endonuclease cleavage patterns, but all were nonneuroinvasive. In addition, all syn+ viruses (23 clones) tested were found to have TK- phenotypes, indicating that the majority of viruses present in the central nervous system were TK- viruses, since about 90% of viruses detected in spinal cords and brains exhibited syn+ phenotypes. These results strongly suggest that the lethal invasion of the central nervous system by HSV-2 TK- and nonneuroinvasive HSV-1 was the consequence of in vivo complementation between the two viruses.     

Functional interaction between herpes simplex virus type 2 gD and HVEM transiently dampens local chemokine production after murine mucosal infection

Herpes virus entry mediator (HVEM) is one of two principal receptors mediating herpes simplex virus (HSV) entry into murine and human cells. It functions naturally as an immune signaling co-receptor, and may participate in enhancing or repressing immune responses depending on the natural ligand used. To investigate whether engagement of HVEM by HSV affects the in vivo response to HSV infection, we generated recombinants of HSV-2(333) that expressed wild-type gD (HSV-2/gD) or mutant gD able to bind to nectin-1 (the other principal entry receptor) but not HVEM. Replication kinetics and yields of the recombinant strains on Vero cells were indistinguishable from those of wild-type HSV-2(333). After intravaginal inoculation with mutant or wild-type virus, adult female C57BL/6 mice developed vaginal lesions and mortality in similar proportions, and mucosal viral titers were similar or lower for mutant strains at different times. Relative to HSV-2/gD, percentages of HSV-specific CD8(+) T-cells were similar or only slightly reduced after infection with the mutant strain HSV-2/gD-Δ7-15, in all tissues up to 9 days after infection. Levels of HSV-specific CD4(+) T-cells five days after infection also did not differ after infection with either strain. Levels of the cytokine IL-6 and of the chemokines CXCL9, CXCL10, and CCL4 were significantly lower in vaginal washes one day after infection with HSV-2/gD compared with HSV-2/gD-Δ7-15. We conclude that the interaction of HSV gD with HVEM may alter early innate events in the murine immune response to infection, without significantly affecting acute mortality, morbidity, or initial T-cell responses after lethal challenge.     

Temperature sensitive mutant of Herpes simplex virus type 1. I. Pathogenicity and immunogenicity]

The aim of the study was to characterize biological features of the sensitive mutant of HSV-1, derived from McIntyre strain by numerous virus passages at lowered replication temperature (28 degrees C). Pathogenicity of obtained ts mutant for inbred mice lines, CFW/Pzh and BALB/cPzh, was determined. Statistically significant decrease in virulence of the mutant for these mouse lines was demonstrated, as compared with the native virus strain, propagated at 37 degrees C. Immunogenic activity of ts mutant of HSV-1 defined by the possibility of mouse protection against infection with high virulent was determined. Mice, which at the time of immunization with ts mutant received Depo-Medrol--an immunosuppressive agent--were also found to be capable of inducing defense mechanisms to infection with the native strain.     

An HSV-1 Vector Expressing Tyrosine Hydroxylase Causes Production and Release of l-DOPA from Cultured Rat Striatal Cells

Abstract: In this report we demonstrate that a defective herpes simplex virus type one (HSV-1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into l -DOPA-producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV-1 vector (pHSVth) and packaged into virus particles using an HSV-1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production of l -DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release of l -DOPA and low levels of dopamine were observed from cells in pHSVth-infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week after infection.     

Disruption of virion host shutoff activity improves the immunogenicity and protective capacity of a replication-incompetent herpes simplex virus type 1 vaccine strain

The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8(-) virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. In this study, a vhs(-)/ICP8(-) HSV-1 mutant was engineered. BALB/c mice were immunized with incremental doses of the vhs(-)/ICP8(-) double mutant or vhs(-) or ICP8(-) single mutants, or the mice were mock immunized, and protective immunity against corneal challenge with virulent HSV-1 was assessed. Mice immunized with the vhs(-)/ICP8(-) mutant showed prechallenge serum immunoglobulin G titers comparable to those immunized with replication-competent vhs(-) virus and exceed those of mice immunized with the ICP8(-) single mutant. Following corneal challenge, the degrees of protection against ocular disease, weight loss, encephalitis, and establishment of latency were similar for vhs(-)/ICP8(-) and vhs(-) virus-vaccinated mice. Moreover, the double deleted vhs(-)/ICP8(-) virus protected mice better in all respects than the single deleted ICP8(-) mutant virus. The data indicate that inactivation of vhs in a replication-incompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.     

Glycosaminoglycans are not indispensable for the anti-herpes simplex virus type 2 activity of lactoferrin

Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In particular, bovine lactoferrin (bLf) has been found to prevent viral infection by binding to heparan sulphate (HS) glycosaminoglycans (GAGs) that in turn can act as cell receptors for human herpetic viruses. In this study we further investigate the mechanism of inhibiting activity of both human lactoferrin (hLf) and bLf against HSV-2. The antiviral effect of these proteins towards HSV-2 strain 333 and its glycoprotein C (gC)-truncated derivative HSV-2 gC-neg1 has been tested in monkey kidney cells. Our results indicate that the antiviral activity of bLf does not involve gC-HS interaction as there was no difference in its effectiveness towards wild type and mutant virus. As regards hLf, the mutant virus HSV-2 gC-neg1 was more sensitive compared to the wild type, suggesting that the human protein might interact with some viral structures that in wild-type viruses are masked by gC. When the modulation of HSV-2 infection by bLf and hLf was investigated under different experimental conditions, the bovine protein proved more effective than the human protein. Moreover, we found that, differently from what observed with HSV-1, bLf inhibited HSV-2 plaque-forming activity also in cells devoid of GAG expression. These results suggest that bLf may block a virus receptor of non-GAG nature and add new information on the anti-herpes virus activity of this protein, confirming it as an outstanding candidate for the treatment of herpetic infections.