Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice

Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for neurovirulence are being used for the treatment of cancer. We have examined the safety of G207, a multimutated replication-competent HSV-1 vector, in mice. BALB/c mice inoculated intracerebrally or intracerebroventricularly with 10(7) PFU of G207 survived for over 20 weeks with no apparent symptoms of disease. In contrast, over 80% of animals inoculated intracerebrally with 1.5 x 10(3) PFU of HSV-1 wild-type strain KOS and 50% of animals inoculated intracerebroventricularly with 10(4) PFU of wild-type strain F died within 10 days. Similarly, after intrahepatic inoculation of G207 (3 x 10(7) PFU) all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 10(6) PFU of KOS. After intracerebroventricular inoculation, LacZ expression was initially observed in the cells lining the ventricles and subarachnoid space; expression decreased until almost absent within 5 days postinfection, with no apparent loss of ependymal cells. G207 DNA could be detected by PCR in the brains of mice 8 weeks after intracerebral inoculation; however, no infectious virus could be detected after 2 days. As a model for latent HSV in the brain, we used survivors of an intracerebral inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high dose of G207 at the same stereotactic coordinates did not result in reactivation of detectable infectious virus or symptoms of disease. We conclude that G207 is safe at or above doses that were efficacious in mouse tumor studies.     

Antiviral activity and dose optimum of recombinant macrophage colony-stimulating factor on herpes simplex genitalis in guinea pigs.

The antiviral activity of recombinant human macrophage CSF (M-CSF) against genital herpes simplex virus type-2 (HSV-2) infection in guinea pigs was investigated. M-CSF stimulates proliferation of human and guinea pig peripheral blood monocytes, specifically the plastic adherent esterase-positive mononuclear cells. When anti-HSV-2 activity of M-CSF was evaluated in guinea pigs by 6 daily injection (s.c.) of M-CSF at various doses (5 x 10(5) to 7 x 10(7) U/kg), we found 2 x 10(6) U/kg to be the optimum dose for protective efficacy against primary HSV-2 infection. Either at a lethal, 5 x 10(5) pfu, or sublethal 5 x 10(4) pfu of virus challenge, animals treated with the optimum regimen of M-CSF exhibited lower herpetic lesion scores (p less than 0.005), and lower mortality (p less than 0.025) than animals in placebo group. M-CSF treatment increased the HSV-infected cell killing activities of plastic-adherent mononuclear cells, indicating that in vivo administration of M-CSF may activate the antiviral effects of guinea pig macrophages that may play a role in protection against severity and mortality of herpetic disease.     

Herpes simplex virus type 2 UL24 gene is a virulence determinant in murine and guinea pig disease models

A herpes simplex virus type 2 (HSV-2) UL24 beta-glucuronidase (UL24-betagluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-betagluc yielded cytopathic effect with syncytium formation. UL24-betagluc replicated to wild-type viral titers in three different cell lines. UL24-betagluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-betagluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-betagluc at a dose equivalent to the LD50 of parental virus (approximately 5 x 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-betagluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.     

Efficacy of oral administration of live herpes simplex virus type 1 as a vaccine.

Mice given herpes simplex virus type 1 (HSV-1) (Miyama +GC strain) intragastrically via a stainless-steel cannula were rendered immune to subsequent lethal intraperitoneal (i.p.) challenge with HSV-1. The orally administered HSV-1 was completely inactivated in the stomach within a few minutes of inoculation. However, systemic immunity was established 14 days after oral inoculation with the virus and retained for up to 6 months. The mechanisms of establishing systemic immunity were investigated by means of adoptive transfer comparisons. When splenic cells from HSV-1-immunized mice were transplanted into nonimmunized mice, all of the recipient mice survived after a lethal i.p. challenge with the virus. Immunity was not established in antithymocyte serum-treated mice or by transfer of serum from immunized to nonimmunized mice. In addition, all HSV-1-immunized mice died after lethal challenge with HSV-2 and influenza virus A. These findings suggest that the immunity was virus specific, with T lymphocytes playing a major role in its establishment. The present study therefore supports the possibility of oral immunization with live HSV-1 as a vaccine.     

Cell-mediated immunity to Friend virus-induced leukemia. II. Characteristics of primary cell-mediated cytotoxic response.

The primary cell-mediated cytotoxic response to a Friend virus-induced leukemia, FBL-3, in C57BL/6 mice was measured by the 125IUdR release assay. Intraperitoneal (i.p.) inoculation of 1 x 10(1) FBL-3 cells produced progressive tumor growth (progressors); subcutaneous (s.c.) inoculation of as many as 5 x 10(6) FBL-3 cells produced only transient tumor growth (regressors), and these mice would subsequently resist i.p. challenge of FBL-3 cells at 3 days after s.c. inoculation. The kinetics of the primary cell-mediated cytotoxic response of regressors was biphasic. Significant cytotoxicity could be detected at 3 to 5 days after s.c. inoculation of 5 x 10(6) FBL-3 cells peaked at days 10 to 14, declined to a very low level or became undetectable around days 20 to 30; then the reactivity reappeared and persisted at least up to 60 days. In progressors, the kinetics of the cell-mediated cytotoxic response was similar to the regressors, but the reactivity was much lower. The cytotoxic response was found to be T cell dependent, during both the first peak (days 10 to 14) and the second peak (days 40 to 60). In adoptive transfer experiments, lymphocytes from regressors gave 90% protection against i.p. challenge of FBL-3; lymphocytes from progressors only gave 40% protection.     

Mechanism of differences in pathogenicity between two variants of a laboratory strain of herpes simplex virus type 1

The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV-1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the -GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated -GCr variant. When the attenuated variant was preinoculated i.p. 24 hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV-1 was shown to be age-dependent.     

Ocular avirulence of a herpes simplex virus type 1 strain is associated with heightened sensitivity to alpha/beta interferon.

BALB/c mice infected on the scarified cornea with herpes simplex virus type 1 strain 35 [HSV-1(35)] rarely developed ocular disease even at challenge doses as high as 10(7) PFU per eye. In contrast, HSV-1(RE) consistently induced stromal keratitis at an inoculum of 2 x 10(4) PFU. The goal of this study was to determine the reason for the difference in virulence between the two HSV strains. Both HSV-1 strains replicated to similar titers in excised corneal "buttons." However, after in vivo infection of the cornea, the growth of strain 35 was evident only during the first 24 h postinfection, whereas the replication of strain RE persisted for at least 4 days. In vitro tests revealed that HSV-1(35) was greater than 10 times more sensitive to alpha/beta interferon (IFN-alpha/beta) than HSV-1(RE). Both strains induced comparable serum levels of IFN after intraperitoneal inoculation. The kinetics of HSV-1(35) clearance from the eye was markedly altered by treatment with rabbit anti-IFN-alpha/beta. Virus titers exceeding 10(4) PFU per eye could be demonstrated 4 to 5 days postinfection in mice given a single inoculation of antiserum 1 h after infection. Furthermore, anti-IFN treatment in 3-week-old mice infected with HSV-1(35) led to the development of clinically apparent corneal disease which subsequently progressed to stromal keratitis in the majority of recipients. These results indicate that the striking difference in the capacity of HSV-1(35) and HSV-1(RE) to induce corneal disease was related to the inherently greater sensitivity of strain 35 to IFN-alpha/beta produced by the host in response to infection.     

Susceptibility of Psammomys obesus and Meriones tristrami to tachyzoites of Neospora caninum

The susceptibility of Psammomys obesus (sand rat) and Meriones tristrami (Tristram's jird) to Neospora caninum was investigated by subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of 10-fold doses of culture-derived tachyzoites. Groups of 5 animals were inoculated with doses of 10-10(7) parasites via each route of inoculation. All but 2 of the sand rats inoculated with doses of 10-10(4) parasites succumbed to the infection by 7-18 days postinfection. All jirds inoculated with 10(7) tachyzoites succumbed by 5-16 days postinfection and those inoculated with 10(6) tachyzoites by 9-25 days. A considerable proportion of the jirds inoculated with 10-10(5) tachyzoites survived. Fibrinous peritonitis with ascites containing numerous tachyzoites was observed in the i.p.-inoculated sand rats and jirds that succumbed to the infection. In the jirds, tachyzoites were also found in pleural exudate. A considerable number (42.8%) of the jirds inoculated s.c. or i.p. exhibited neuromuscular symptoms, expressed in ataxia, head tilt, circling movement, and posterior paralysis. Seven successive passage of tachyzoites were achieved in sand rats with doses of 10(5) parasites and in jirds with doses of 10(7) parasites. All surviving jirds became seroconverted and were immune to lethal challenge.     

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.     

Delayed-Type Hypersensitivity (DTH) Response to Dengue Virus in Mice: Effect of Route of Sensitization and Splenectomy

This study was designed to determine the role of the sensitization route and the spleen in the development of delayed-type hypersensitivity (DTH) to dengue virus in mice. DTH was measured by footpad swelling response. Strong but transient DTH was produced in cyclophosphamide (CY) pretreated mice sensitized subcutaneously (s.c.) or intravenously (i.v.) with dengue virus type 4. Subcutaneous inoculation of virus in incomplete Freund's adjuvant (IFA) further enhanced the DTH elicited. The time course of DTH generated by s.c. and i.v. sensitization were similar with the peak reactivity seen on day 6 after sensitization. Poor DTH was observed in mice given an i.p. inoculation even when CY and/or IFA were used. Intracerebral (i.c.) inoculation also sensitized mice poorly. Splenectomized mice showed enhanced DTH response when compared to intact mice. In contrast to intact mice, pretreatment of splenectomized mice with CY did not alter the DTH level. Splenectomized mice inoculated s.c. with virus in IFA showed poorer DTH than mice sensitized with virus alone.     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

Susceptibility of common voles to experimental toxoplasmosis

Common voles (Microtus arvalis) in groups of nine to 10 animals were inoculated per os with a dose of 1, 10, 1x10(2), 1x10(3), and of the K1 strain of Toxoplasma gondii. All the common voles inoculated with 1 to 1 x 10(3) oocysts remained subclinical and survived. Three of the 10 voles inoculated with 1 x 10(4) oocysts died between days 7 and 12 post inoculation (p.i.). Antibodies were demonstrated in all the infected voles killed on day 60 p.i. The highest antibody titres in voles detected by the dye test (DT) and latex agglutination test (LAT) were 1,024 and 1,280, respectively.     

Expression of human immunodeficiency virus type 1 gp120 from herpes simplex virus type 1-derived amplicons results in potent, specific, and durable cellular and humoral immune responses

Herpes simplex virus type 1 (HSV-1) infects a wide range of cells, including dendritic cells. Consequently, HSV-1 vectors may be capable of eliciting strong immune responses to vectored antigens. To test this hypothesis, an HSV-1 amplicon plasmid encoding human immunodeficiency virus type 1 gp120 was constructed, and murine immune responses to helper virus-free amplicon preparations derived from this construct were evaluated. Initial studies revealed that a single intramuscular (i.m.) injection of 10(6) infectious units (i.u.) of HSV:gp120 amplicon particles (HSV:gp120) elicited Env-specific cellular and humoral immune responses. A potent, CD8(+)-T-cell-mediated response to an H-2D(d)-restricted peptide from gp120 (RGPGRAFVTI) was measured by a gamma interferon ELISPOT and was confirmed by standard cytotoxic-T-lymphocyte assays. Immunoglobulin G enzyme-linked immunosorbent assay analysis showed the induction of a strong, Env-specific antibody response. An i.m. or an intradermal administration of HSV:gp120 at the tail base elicited a more potent cellular immune response than did an intraperitoneal (i.p.) inoculation, although an i.p. introduction generated a stronger humoral response. The immune response to HSV:gp120 was durable, with robust cellular and humoral responses persisting at 171 days after a single 10(6)-i.u. inoculation. The immune response to HSV:gp120 was also found to be dose dependent: as few as 10(4) i.u. elicited a strong T-cell response. Finally, HSV:gp120 elicited significant Env-specific cellular immune responses even in animals that had been previously infected with wild-type HSV-1. Taken together, these data strongly support the use of helper-free HSV-1 amplicon particles as vaccine delivery vectors.     

The hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle-inoculated, cultured organisms.

The human immune response to natural infection with Borrelia burgdorferi appears to differ from that seen in small mammals infected by needle inoculation. In humans, antibody to outer surface proteins A and B (OspA and OspB) is not detectable until late in infection, but small mammals inoculated with B. burgdorferi produce early antibody to OspA and OspB. To investigate this disparity we compared the immune response in hamsters to B. burgdorferi after needle inoculation with cultured organisms or infected tick homogenates with the immune response after tick transmitted (natural) infection. We determined that the antibody response to OspA and OspB after natural infection of hamsters is similar to that seen in humans, and differs from the antibody response after hamster infection by needle inoculation. High titers of antibody to OspA and OspB were undetectable even 42 wk after bite by B. burgdorferi-infected ticks. The failure to produce antibody to OspA and OspB was not dependent on challenge dose, because animals inoculated by needle with low doses (1 x 10(5) to 1 x 10(6) cells) of B. burgdorferi produced antibody to OspA and OspB. A rapid but limited anti-41-kDa response was observed. One possible new Ag, 43 kDa (p43), was identified. The antibody response to p43 was independent of the route of inoculation. Our results suggest that the hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle inoculated, cultured organisms.     

The transneuronal spread phenotype of herpes simplex virus type 1 infection of the mouse hind footpad.

The mouse hind footpad inoculation model has served as a standard laboratory system for the study of the neuropathogenesis of herpes simplex virus type 1 (HSV-1) infection. The temporal and spatial distribution of viral antigen, known as the transneuronal spread phenotype, has not previously been described; nor is it understood why mice develop paralysis in an infection that involves sensory nerves. The HSV-as-transneuronal-tracer experimental paradigm was used to define the transneuronal spread of HSV-1 in this model. A new decalcification technique and standard immunocytochemical staining of HSV-1 antigens enabled a detailed analysis of the time-space distribution of HSV-1 in the intact spinal column. Mice were examined on days 3, 4, 5, and 6 postinoculation (p.i.) of a lethal dose of wild-type HSV-1 strain 17 syn+. Viral antigen was traced retrograde into first-order neurons in dorsal root ganglia on day 3 p.i., to the dorsal spinal roots on days 4 and 5 p.i., and to second- and third-order neurons within sensory regions of the spinal cord on days 5 and 6 p.i. HSV-1 antigen distribution was localized to the somatotopic representation of the footpad dermatome within the dorsal root ganglia and spinal cord. Antigen was found in the spinal cord gray and white matter sensory neuronal circuits of nociception (the spinothalamic tract) and proprioception (the dorsal spinocerebellar tract and gracile fasciculus). Within the brain stems and brains of three paralyzed animals examined late in infection (days 5 and 6 p.i.), HSV antigen was restricted to the nucleus subcoeruleus region bilaterally. Since motor neurons were not directly involved, we postulate that hindlimb paralysis may have resulted from intense involvement of the posterior column (gracile fasciculus) in the thoracolumbar spinal cord, a region known to contain the corticospinal tract in rodents.     

Suppressive effect of adrenalectomy on growth of L1210 leukemic cells in ascites

This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF1 mice. Varying doses of 1.5 x 10(4), 5.0 x 10(5), and 1.5 x 10(6) viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham-operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1.5 x 10(4) or 5.0 x 10(5) tumour cells had a smaller number of tumour cells in ascites than sham-operated controls. However, after inoculation of 1.5 x 10(6) cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham-operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 micrograms dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using [125I]-iododeoxyuridine-labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5.0 x 10(5) labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham-operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time.     

Antiviral activity of a thymic factor in experimental viral infections. I. Thymic hormonal effect on survival, interferon production and NK cell activity in Mengo virus-infected mice

A partially purified thymic factor, thymostimulin (TS), significantly increased the survival rate of adult, immune-intact mice infected with the neurotropic Mengo virus. TS treatment was begun after virus inoculation by daily i.p. injections. In untreated C57BL/6 mice, LD50 was reached with 1 X 10(4) PFU, but 10-fold more virus (i.e., 1 X 10(5) PFU) was needed to reach LD50 in TS-treated animals. TS effect on survival, though, could be observed with several virus doses (1 X 10(3) to 1 X 10(6) PFU) (p less than 0.001). A significant effect on survival was also observed with outbred ICR mice (p less than 0.005). Serum interferon (IFN) levels in the Mengo virus-infected mice were relatively low (average peak 300 U/ml), but were significantly increased (two- to ninefold) in the TS-treated mice. Peak serum levels were reached earlier in TS than in control animals (24 hr and 72 hr, respectively). Both acid-labile and acid-stable type I IFN production were augmented by TS in the Mengo virus-infected mice. Natural killer activity was also enhanced by TS, in particular on the second day after virus inoculation. In addition, MP-virus was used as a second, unrelated virus challenge. This virus caused a nonlethal infection, with relatively high levels of serum IFN (average peak 10,000 U/ml). TS increased IFN levels (two- to eight-fold) also in this challenge system. In conclusion, TS causes a nonspecific enhancement of endogenous production of IFN and has a significant effect on the survival of lethally infected mice. The data indicate a potential application of thymic factors for the treatment of viral infections.