Monoclonal antibodies suppress replication of herpes simplex virus type 1 in trigeminal ganglia.

The effect of monoclonal antibodies on the growth of herpes simplex virus type 1 in trigeminal ganglia was investigated. Four-week-old mice were infected on an abrased cornea with herpes simplex virus type 1. Forty-eight hours after infection, trigeminal ganglia ipsilateral with infected eyes were removed and placed in culture. Incubation of infected ganglia in the presence of a pool of nonneutralizing monoclonal antibodies specific for glycoproteins of gB and gE suppressed virus growth by greater than 90%. This was comparable to the amount of suppression observed when infected ganglia were incubated in hyperimmune serum. Individual monoclonal antibodies were less efficient, being able to inhibit virus growth by only two- to threefold. The mechanism of suppression was examined. Reduction in virus growth was observed under conditions in which all susceptible ganglion cells were infected in vitro before nonneutralizing monoclonal antibody was added. Similar results were obtained in tests with virus-infected neuroblastoma cells. Furthermore, suppression of infectious progeny was seen in the absence of complement and immunologically reactive cells. Thus, neither virus neutralization nor immunocytolysis could account for the effects of antibody on virus growth. Rather, the data suggest that antibody can bind to herpes simplex virus type 1-infected neuronal cells and suppress intracellular virus replication.     

Effects of Nerve Growth Factor and Phorbol Derivative on Reactivation of Herpes Simplex Virus Type 1 in Cultured Cells of Latently Infected Adult Mouse Trigeminal Ganglia

Reactivation of herpes simplex virus type 1 (HSV-1) occurred rapidly in cells of latently infected adult mouse trigeminal ganglia which were cultured in serum-free medium in the presence of sufficient nerve growth factor (NGF). However, HSV-1 reactivation was delayed significantly in ganglionic cultures in the absence of exogenous NGF or in cultures treated with 2-aminopurine in the presence of NGF. The delayed viral reactivation in ganglionic cultures without NGF was accelerated by treatment with phorbol myristate acetate or dibutyryl cyclic AMP. Culture conditions which affected HSV-1 reactivation did not affect replication of HSV-1 in normal ganglionic cultures.     

Adaptive and innate transforming growth factor beta signaling impact herpes simplex virus 1 latency and reactivation

Innate and adaptive immunity play important protective roles by combating herpes simplex virus 1 (HSV-1) infection. Transforming growth factor β (TGF-β) is a key negative cytokine regulator of both innate and adaptive immune responses. Yet, it is unknown whether TGF-β signaling in either immune compartment impacts HSV-1 replication and latency. We undertook genetic approaches to address these issues by infecting two different dominant negative TGF-β receptor type II transgenic mouse lines. These mice have specific TGF-β signaling blockades in either T cells or innate cells. Mice were ocularly infected with HSV-1 to evaluate the effects of restricted innate or adaptive TGF-β signaling during acute and latent infections. Limiting innate cell but not T cell TGF-β signaling reduced virus replication in the eyes of infected mice. On the other hand, blocking TGF-β signaling in either innate cells or T cells resulted in decreased latency in the trigeminal ganglia of infected mice. Furthermore, inhibiting TGF-β signaling in T cells reduced cell lysis and leukocyte infiltration in corneas and trigeminal ganglia during primary HSV-1 infection of mice. These findings strongly suggest that TGF-β signaling, which generally functions to dampen immune responses, results in increased HSV-1 latency.     

A herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia.

Vmw65, a herpes simplex virus type 1 (HSV-1) tegument protein, in association with cellular proteins, transactivates viral immediate early genes. In order to examine the role of Vmw65 during acute and latent infection in vivo, a mutant virus (in1814), containing a 12-base-pair insertion in the Vmw65 gene, which lacks the transactivating function of Vmw65 (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989) was examined in mice. Following corneal inoculation, the parental virus (17+) and the revertant (1814R) replicated effectively in eyes and trigeminal ganglia with 30 to 60% mortality. At either equal PFU or equal particle numbers, in1814 did not replicate in trigeminal ganglia and none of the infected mice died. Although in1814 did not replicate following corneal inoculation, it established latent infection in trigeminal ganglia. HSV-1 in1814 reactivated at explant as efficiently and rapidly as did 17+ and 1814R. Even low amounts of inoculated in1814 (10(2) PFU) were sufficient to establish latent infection in some animals. Since infectious in1814 was not detected at any time in mouse trigeminal ganglia, in1814 provided a unique opportunity to determine how soon after primary infection latency begins. Latent in1814 infection was detected shortly after virus reached the sensory ganglia, between 24 to 48 h postinfection. Thus, though Vmw65 may be required for lytic infection in vivo, it is dispensable for the establishment of and reactivation from latent infection. These data support the hypotheses that the latent and lytic pathways of HSV-1 are distinct and that latency is established soon after infection without a requirement for viral replication. However, the levels of Vmw65 reaching neuronal nuclei may be a critical determinant of whether HSV-1 forms a lytic or latent infection.     

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3-5 days after infection, and is undetectable by 7-10 days. During the period of HSV-1 replication, macrophages and gammadelta TCR+ T lymphocytes infiltrate the TG, and TNF-alpha, IFN-gamma, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-alpha, IFN-gamma, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and gammadelta TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-alpha and iNOS, whereas gammadelta TCR+ T cells produce IFN-gamma. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-alpha or IFN-gamma all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-alpha or IFN-gamma markedly reduced the accumulation of both macrophages and gammadelta TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and gammadelta TCR+ T lymphocytes through their production of antiviral molecules TNF-alpha, NO, and IFN-gamma. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.     

Alpha/Beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type 1

In vivo evidence suggests that T-cell-derived gamma interferon (IFN-gamma) can directly inhibit the replication of herpes simplex virus type 1 (HSV-1). However, IFN-gamma is a weak inhibitor of HSV-1 replication in vitro. We have found that IFN-gamma synergizes with the innate IFNs (IFN-alpha and -beta) to potently inhibit HSV-1 replication in vitro and in vivo. Treatment of Vero cells with either IFN-beta or IFN-gamma inhibits HSV-1 replication by <20-fold, whereas treatment with both IFN-beta and IFN-gamma inhibits HSV-1 replication by approximately 1,000-fold. Treatment with IFN-beta and IFN-gamma does not prevent HSV-1 entry into Vero cells, and the inhibitory effect can be overcome by increasing the multiplicity of HSV-1 infection. The capacity of IFN-beta and IFN-gamma to synergistically inhibit HSV-1 replication is not virus strain specific and has been observed in three different cell types. For two of the three virus strains tested, IFN-beta and IFN-gamma inhibit HSV-1 replication with a potency that approaches that achieved by a high dose of acyclovir. Pretreatment of mouse eyes with IFN-beta and IFN-gamma reduces HSV-1 replication to nearly undetectable levels, prevents the development of disease, and reduces the latent HSV-1 genome load per trigeminal ganglion by approximately 200-fold. Thus, simultaneous activation of IFN-alpha/beta receptors and IFN-gamma receptors appears to render cells highly resistant to the replication of HSV-1. Because IFN-alpha or IFN-beta is produced by most cells as an innate response to virus infection, the results imply that IFN-gamma secreted by T cells may provide a critical second signal that potently inhibits HSV-1 replication in vivo.     

The dominant-negative herpes simplex virus type 1 (HSV-1) recombinant CJ83193 can serve as an effective vaccine against wild-type HSV-1 infection in mice

By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral alpha, beta, and gamma1 genes but little or no gamma2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.     

Reactivation of Herpes Simplex Virus Type 1 in Cells of Latently Infected Mouse Trigeminal Ganglia Cultured in Serum-Free Medium

Herpes simplex virus type 1 (HSV-1) was reactivated more rapidly in cells of latently infected mouse trigeminal ganglia which were cultured in serum-free medium (after 3.7 days of cultivation) than in those cultured in serum-containing Dulbecco's modified Eagle's medium (after 8.5 days of cultivation). The concentration of calcium ion (Ca²⁺) in the medium affected HSV-1 reactivation in ganglionic cultures, and 0.9 mM was the optimum concentration for the reactivation. Reactivation was delayed significantly in ganglia put into culture 4 months or more after infection compared with those cultured 1 month after infection.