Localization of a Passively Transferred Human Recombinant Monoclonal Antibody to Herpes Simplex Virus Glycoprotein D to Infected Nerve Fibers and Sensory Neurons In Vivo

A human recombinant monoclonal antibody to herpes simplex virus (HSV) glycoprotein D labeled with the fluorescent dye Cy5 was administered to mice infected in the cornea with HSV type 1 (HSV-1). The distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. The antibody was detected on HSV-infected nerve fibers in the cornea--identified by colocalization with HSV antigens and the neuritic markers neurofilament, GAP-43, synapsin-1, and CNPase--and on the perikarya of sensory neurons in the HSV-1-infected neurons in ipsilateral trigeminal ganglia. Antibodies have been shown to be effective against many neurotropic viruses, often in the absence of obvious cell damage. Observations from experimental HSV infections suggest that antibodies could act in part by interfering with virus expression in the ganglia and/or with axonal spread. The present results provide morphological evidence of the localization of antiviral antibodies at anatomical sites relevant to such putative antibody-mediated protective actions and suggest that viral glycoproteins are accessible to antibodies on infected nerve fibers and sensory neurons.     

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.     

Pathogenesis of herpes simplex virus type 2 virion host shutoff (vhs) mutants

During lytic infection, the virion host shutoff (vhs) protein mediates the rapid degradation of mRNA and the shutoff of host protein synthesis. In vivo, herpes simplex virus type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. Homologs of vhs exist in all of the neurotropic herpesviruses, and the goal of this study was to determine the virulence of HSV-2 mutants lacking vhs. Two HSV-2 recombinants were used in this study: 333-vhsB, which has a lacZ cassette inserted into the N terminus of vhs, and 333d41, which has a 939-bp deletion in vhs. As expected, both 333-vhsB and 333d41 failed to induce the cellular RNA degradation characteristic of HSV. Corneal, vaginal, and intracerebral routes of infection were used to study pathogenesis. Both viruses grew to significantly lower titers in the corneas, trigeminal ganglia, vaginas, dorsal root ganglia, spinal cords, and brains of mice than wild-type and rescue viruses, with a correspondingly reduced induction of disease. Both viruses, however, reactivated efficiently from explanted trigeminal ganglia, showing that vhs is dispensable for reactivation. The lethality of 333d41 following peripheral infection of mice, however, was significantly higher than that of 333-vhsB, suggesting that some of the attenuation of 333-vhsB may be due to the presence of a lacZ cassette in the vhs locus. Taken together, these data show that vhs represents an important determinant of HSV-2 pathogenesis and have implications for the design of HSV-2 recombinants and vaccines.     

Encephalitis resulting from reactivation of latent herpes simplex virus in mice.

Herpes simplex virus (HSV) encephalitis was produced in mice from reactivation of latent virus. Two experimental models were used: the trigeminal model after corneal inoculation of HSV, and the hypoglossal model after tongue inoculation of HSV. In the trigeminal model, cyclophosphamide treatment induced reactivation of latent virus in ganglia but not in central nervous system tissue. Spread of the reactivated virus from ganglia to brain occurred only in mice deficient in anti-HSV antibody. In the hypoglossal model, sectioning of the hypoglossal nerve provoked chromatolysis in the corresponding central nervous system motor neurons and occasionally reactivated latent HSV in the brains of mice. These results suggest that HSV encephalitis can result from the spread of reactivated virus from ganglia to brain and also from in situ reactivation in brain.     

Herpes simplex virus latency in the rabbit trigeminal ganglia: ganglionic superinfection

It has been confirmed and further documented that infection of the rabbit cornea with the E-43 strain of HSV-1 precludes superinfection of the corresponding trigeminal ganglia by another HSV strain, i.e., the challenging virus does not establish latency and can not be recovered from the ganglia. It was shown that after primary infection, a state of resistance is established in the neuronal cells of the ganglia, and although the challenging strain reaches the ganglia, it does not cause discernible acute infection, and does not displace the resident virus in the ganglia. This protection was present 6 months after primary infection, was independent of immune factors such as circulating or secretory antibodies, and was localized to the point of entry of the primary infecting strain and the sensory neurons that innervate that site. The smallest inoculum that provided protection from ganglionic superinfection was that which produced overt disease in the eye, although different degrees of disease resulted from varying inocula above this minimum. Asymptomatic primary infections produced by subminimal inocula of the E-43 strain or by the HSV recombinant strain, F(MP)F, which is avirulent for the rabbit eye, protected against severe disease and death, but the degree of protection against ganglionic superinfection was variable and depended on the time of challenge. These findings suggest that susceptible neurons in the trigeminal ganglion, when "occupied" by an infecting strain, cannot be superinfected by a second strain.     

Medroxyprogesterone acetate inhibits CD8+ T cell viral-specific effector function and induces herpes simplex virus type 1 reactivation

Clinical research suggests hormonal contraceptive use is associated with increased frequencies of HSV reactivation and shedding. We examined the effects of medroxyprogesterone acetate (MPA), the compound most commonly used for injectable hormonal contraception, on HSV type 1 (HSV-1) reactivation and CD8(+) T cell function in murine trigeminal ganglia (TG). In ex vivo TG cultures, MPA dramatically inhibited canonical CD8(+) T cell effector functions, including IFN-gamma production and lytic granule release, and increased HSV-1 reactivation from latency. In vivo, MPA treatment of latently infected ovariectomized mice inhibited IFN-gamma production and lytic granule release by TG resident CD8(+) T cells stimulated directly ex vivo. RNA specific for the essential immediate early viral gene ICP4 as well as viral genome DNA copy number were increased in mice that received MPA during latency, suggesting that treatment increased in vivo reactivation. The increase in HSV-1 copy number appeared to be the result of a two-tine effect, as MPA induced higher reactivation frequencies from latently infected explanted TG neurons in the presence or absence of CD45(+) cells. Our data suggest hormonal contraceptives that contain MPA may promote increased frequency of HSV reactivation from latency through the combinatory effects of inhibiting protective CD8(+) T cell responses and by a leukocyte-independent effect on infected neurons.     

Eradication of herpes simplex virus persistence in rat trigeminal ganglia by retrograde axoplasmic transport.

Potential use of retrograde axoplasmic flow to eradicate virus persistence in ganglionic cells was studied in a new herpes simplex virus (HSV) persistence model in rat trigeminal ganglia. After injection of the F strain of HSV type 1 into the mental nerve, viral antigens were detectable in the ganglia by the immunofluorescence and peroxidase methods between postinoculation (p.i.) days 3 and 6 but not thereafter. None of 82 inoculated rats showed signs of acute illness, and some survived for more than 502 days without symptoms. By cocultivation of ganglion tissues with Vero cells, the virus was isolated from 42 of 49 ganglia (85.7%) between 15 and 386 days (p.i.). HSV DNA was solely localized in the nucleus of neurons by immunoperoxidase staining of paraffin sections with a biotinylated HSV DNA probe, and the presence of HSV DNA-positive cells was confirmed in four of four ganglia on p.i. day 6 and in five of six on p.i. day 502. The efficacy of axoplasmic flow in drug delivery to ganglionic cells was investigated by injection of doxorubicin (ADM) into the nerve once used for virus inoculation. As early as 19 h after injection, strong ADM-specific autofluorescence was seen in the nuclei of neurons parental to the mental nerve and in those of adjacent Schwann cells, and the death of ADM-positive cells subsequently ensued. A single injection of ADM reduced the virus isolation rate from 31/37 (84%) to 3/37 (8%).     

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7-8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10-13 microns) VIP-IR cells and large (25-32 microns) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.     

Trigeminal ganglion infection by thymidine kinase-negative mutants of herpes simplex virus after in vivo complementation.

Infection of trigeminal ganglion by herpes simplex virus (HSV) thymidine kinase-negative (TK-) mutants was investigated in mixed infection studies in mice. Mice were corneally inoculated with TK- HSV alone or with mixtures of TK- HSV-TK+ HSV. When inoculated alone, an arabinosylthymine-selected HSV type 1 TK- mutant and a HSV type 2 TK- deletion mutant infected mouse ocular tissues but rarely infected ganglion tissues. However, both TK- mutants readily infected ganglion tissues when they were inoculated in mixtures with TK+ HSV. By means of mixed infection studies, it was demonstrated that TK- HSV could readily establish acute and latent ganglion infections. It was thought that the frequent infection of trigeminal ganglion tissue by both TK- mutants after mixed TK(-)-TK+ HSV infection was the result of in vivo complementation. After mixed TK(-)-TK+ HSV infection and subsequent cultivation of ganglion explants in arabinosylthymine, results supported the conclusion that when TK- was present in ganglia it was in the same neurons that contained TK+ HSV.     

Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.