Relative role of B and T lymphocytes in pathogenesis of a murine herpes virus.

Pathogenesis of a murine herpes virus was investigated in inbred strains (BALB/c, CBA, AKR and C57BL/10) of mice. After intranasal inhalation, virus was found to replicate primarily in the lungs, followed by haematogenous spread to the target organs (adrenal glands and ganglia). AKR (H-2k) were found to be most susceptible to virus infection while CBA (H-2k) mice appeared to be relatively resistant. Infection of B-cell depleted BALB/c mice resulted in detection of lower lung virus titres in B-cell depleted animals as compared to normal intact mice. Moreover, 3 of 12 normal mice in untreated group died of virus infection while deaths did not occur in the B-cell depleted group. Results of T-cell subset depletion experiments in BALB/c mice revealed maximum mortality in the group depleted of both Lyt-2+ and L3T4+ subpopulations. Infectious virus titres were also higher in lungs of T-cell depleted animals.

Herpes simplex virus type 2 latency in the footpad of mice: effect of acycloguanosine on the recovery of virus.

Herpes simplex virus type 2 has been reactivated from the latent state in the footpad and dorsal root ganglia of acycloguanosine-treated BALB/c mice. Virus was also recovered from the footpad tissue but not from the ganglia of denervated, latently infected mice. Treatment in vitro of explanted footpad cultures with acycloguanosine or phosphonoacetic acid did not affect the rate of virus reactivation. In all the isolates examined the virus was found to be acycloguanosine-sensitive. Recovery of virus from footpad tissue of mice after a long period of acycloguanosine treatment supports the theory that virus had been truly latent in the footpad and not in a state of persistent infection.

Definition of a virulence-related antigen of Neisseria gonorrhoeae with monoclonal antibodies and lectins.

Variants of one strain of Neisseria gonorrhoeae, grown in vivo or in vitro, that have been previously shown to differ in infectivity, serum resistance, and capsule production were compared with use of monoclonal antibodies and lectins. Monoclonal antibodies to virulent gonococci recognized an antigenic site of the lipopolysaccharide (LPS) produced in large amounts by gonococci grown in vivo but present only in a small proportion of in vitro-grown gonococci. This antigen (C-LPS) was found in all 85 different gonococcal isolates studied but not among nonpathogenic neisseriae. It was shared by group B and C meningococci but not by groups A and D. Enzyme-linked immunosorbent assay and Western blot analysis showed that N-acetylglucosamine and N-acetylgalactosamine form part of the epitope. The C-LPS antigen was shown by immunofluorescence to be present on the surface of the gonococci and also free as slime. This antigen appears to confer resistance to killing by normal sera.

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations.

Clonal analysis of T-cell responses to herpes simplex virus: isolation, characterization and antiviral properties of an antigen-specific helper T-cell clone.

A herpes simplex virus (HSV)-specific long-term T-cell clone has been established from the draining lymph node cells of BALB/c mice; the cells required repeated in vitro restimulation with UV-irradiated virus. The established T-cell clone expresses the Thy-1 and Lyt-1+2,3- surface antigens. For optimal proliferation of the cloned cells, both the presence of specific antigen and an exogenous source of T-cell growth factor are required. The proliferative response of the cloned T cells was found to be virus-specific but it did not distinguish between HSV-1 and HSV-2. Adoptive cell transfer of the cloned T cells helped primed B cells to produce anti-herpes antibodies: the response was antigen-specific and cell dose-dependent. The clone failed to produce a significant DTH reaction in vivo, but did produce high levels of macrophage-activating factor. Furthermore, the T-cell clone could protect from HSV infection, as measured by a reduction in local virus growth, and by enhanced survival following the challenge of mice with a lethal dose of virus. The mechanism(s) whereby this clone protects in vivo is discussed.

T cell-macrophage interaction in arginase-mediated resistance to herpes simplex virus.

Peritoneal macrophages activated by-products derived from a herpes simplex virus-specific helper T cell clone were used to investigate intrinsic and extrinsic resistance mechanisms to herpes simplex virus type 1 infection in vitro. T cell-activated macrophages produced fewer infective centres, indicating enhanced intrinsic resistance, and markedly reduced the growth of virus in a permissive cell line. The reduction in virus growth correlated with the depletion of arginine in the support medium, presumably resulting from increased arginase production by activated macrophages. The significance of these findings for antiviral immunity in vivo is discussed.

The use of monoclonal antibodies in (reverse) passive haemagglutination tests for herpes simplex virus antigens and antibodies.

Three monoclonal antibodies against herpes simplex virus type 2 have been tested for their suitability as reagents in reverse passive haemagglutination. Two of these antibodies with specificity for virus glycoprotein D, when linked to red blood cells, were able to capture antigens without being agglutinated, but addition of immune serum subsequently led to agglutination. Haemagglutination using these monoclonal antibody-linked, antigen-captured red cells was readily applicable to testing human sera for antibodies to herpes simplex virus and the titres obtained correlated with those from virus plaque neutralisation tests. The procedure has been termed "Specific Antigen Capture Passive Haemagglutination." A further monoclonal antibody with specificity for the major DNA-binding protein of type 2 herpes virus-infected cells (a nonstructural protein) showed conventional reverse passive haemagglutination when linked to red blood cells and was specific for type 2 herpes simplex virus. The nature and potential uses of these simple reverse passive haemagglutination procedures using monoclonal antibody reagents are discussed.

Role of neutralizing antibodies and T-cells in pathogenesis of herpes simplex virus infection in congenitally athymic mice.

Congenitally athymic nude mice were infected with 10(4) p.f.u. herpes simplex type 1 (strain SC16). Following the passive transfer of neutralizing monoclonal antibodies (AP7, AP8 and AP12) it was observed that AP7 alone reduced the virus infectivity in the nervous system; AP8 and AP12 failed to protect mice probably due to poor in vivo binding to the neutralization site on the virus. Latent ganglionic infection could be established in nude mice following adoptive transfer of optimum number (2 x 10(7) cells/mouse) of immune lymph node cells from day 7 herpes virus-infected hairy immunocompetent donor mice. Moreover, in some of the immune lymph node cell protected nudes, latency could be maintained even in complete absence of neutralizing antibodies. Results of ear-ablation experiments revealed that removal of primary source of infection after day 5 of infection reduced the amount of virus in the ganglia and spinal cord. Acute neurological infection was not detected following transfer of protective anti-gp-D neutralizing antibody (LP2) in combination with removal of infected pinna. These data suggest that continuous seeding of virus occurs in related ganglia via the axonal route from infected ear pinna. It appears that local T-cell-mediated immune mechanisms are involved in maintenance of latency.

Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase?

Proteose-peptone-activated mouse macrophages can prevent productive infection by herpes simplex virus in neighboring cells in vitro whether or not those cells belong to the same animal species. The effect does not require contact between the macrophages and the infected cells, may be prevented by adding extra arginine to the medium, and may be reversed when extra arginine is added 24 h after the macrophages. Arginase activity was found both intracellularly and released from the macrophages. The extracellular enzyme is quite stable; 64% activity was found after 48 h of incubation at 37 degrees C in tissue culture medium. No evidence was found that the inefficiency of virus replication in macrophages was due to self-starvation by arginase. As might be predicted macrophages can, by the same mechanism, limit productive infection by vaccinia virus.

Pathogenesis of herpes simplex virus in B cell-suppressed mice: the relative roles of cell-mediated and humoral immunity.

B cell responses of Balb/c mice were suppressed using sheep anti-mouse IgM serum. At 4 weeks, both B cell-suppressed and normal littermates were infected in the ear pinna with herpes simplex virus type 1 (HSV-1). The B cell-suppressed mice failed to produce neutralizing herpes antibodies in their sera but had a normal cell-mediated immunity (CMI) response as measured by a delayed hypersensitivity skin test. Although the infection was eliminated from the ear in both B cell-suppressed and normal mice by day 10 after infection, there was an indication that B cell-suppressed mice had a more florid primary infection of the peripheral and central nervous system and also a higher incidence of a latent infection. These results support the hypothesis that antibody is important in restricting the spread of virus to the central nervous system, whereas CMI is important in clearing the primary infection in the ear pinna.

Pathogenesis of herpes simplex virus in congenitally athymic mice: the relative roles of cell-mediated and humoral immunity.

Athymic nude (nu/nu) mice were inoculated in the ear pinna with 10(4) p.f.u. herpes simplex virus type 1 (strain SC 16). Initially, the virus was observed to replicate in the pinna, spreading via a neurological route to the dorsal root ganglia, spinal cord, brain and adrenal glands. Following the transfer of lymphoid cells from day 7 herpesvirus-infected hairy immunocompetent donors into infected nude mice, virus was not isolated from the pinna and nervous system of the majority of the mice. The passive transfer of neutralizing polyclonal anti-herpesvirus serum or neutralizing monoclonal anti-gp D serum did not reduce infectivity in the pinna, but markedly reduced the amount of virus in the ganglia and spinal cord. These data suggest that neutralizing antibodies play an important role in restricting the movement of virus to the nervous system, whereas cell-mediated immune (CMI) mechanisms are essential for eliminating virus from the pinna.

In vitro stimulation of rabbit T lymphocytes by cells expressing herpes simplex antigens.

Lymphocyte stimulation responses to herpes antigens were studied using virus-infected X-irradiated cells. Rabbits were immunized with herpes simplex virus type 1 (strain HFEM) grown in RK 13 cells. For in vitro stimulation assay BHK21 cells were X-irradiated (15 000 rad) and infected with a high m.o.i. of a temperature-sensitive (ts) mutant (N102) of HFEM strain at the non-permissive temperature (38.5 degrees C) of virus. Virus antigens were expressed on the infected cells and there was no leakage of infectious virus into the medium at 38.5 degrees C. T lymphocytes from rabbits immunized with herpes simplex virus were specifically activated by herpesvirus-infected X-irradiated cells; lymph node cells from rabbits immunized with RK13 cells and from non-immune rabbits showed no proliferative response.

Atypical patterns of neural infection produced in mice by drug-resistant strains of herpes simplex virus.

Mice inoculated intracerebrally (i.c.) with a mutant strain of HSV were found to develop cataracts 1 to 2 months after inoculation. Cataract formation was subsequently shown to follow an acute retinitis which commenced within 1 week of inoculation. The mutant had been selected for high resistance to the nucleoside analogue acyclovir and has been shown previously to be defective in the induction of thymidine kinase and also to express an altered DNA polymerase. The LD50 for mice inoculated i.c. was greater than 10(5) p.f.u. compared with approx 7 p.f.u. for the parental strain. Studies of virus replication following i.c. inoculation with a sublethal dose of the mutant revealed that only small amounts of infectious virus were produced in the brain, but during a period from 6 to 12 days after inoculation vigorous replication occurred in retinal tissue, producing very high titres of virus.

Tolerance and immunity in mice infected with herpes simplex virus: studies on the mechanism of tolerance to delayed-type hypersensitivity.

Tolerance to delayed-type hypersensitivity is produced in mice following an intravenous injection of herpes simplex virus. This form of tolerance is produced early on, following simultaneous injections of virus subcutaneously and intravenously, and is long lasting (greater than 100 days). The early tolerance mechanism is resistant to high doses of cyclophosphamide and is not transferable by serum or spleen cells taken after 7 days. However, spleen cells taken at 14 days onwards inhibit the induction of delayed hypersensitivity when transferred to normal syngeneic recipients. These cells are T lymphocytes and are specific for the herpes type used in the induction.