Production of soluble suppressor factor by spleen cells from mice immunized with type III pneumococcal polysaccharide

Supernatant fluid (SF) derived from spleen cell cultures, obtained from mice 16 hr after immunization with 0.5 microgram of Type III pneumococcal polysaccharide (SSS-III), suppressed the antibody response when SF was given (i.v.) 3 hr before immunization with SSS-III. Such suppression was antigen specific and could be reproduced by SF derived from cultures of T cells from mice immunized with SSS-III (0.5 microgram) or by SF derived from cultures of spleen cells from mice primed with a subimmunogenic dose of SSS-III (0.005 microgram). Adsorption of SF with SSS-III covalently bound to a Sepharose 4B column did not alter the ability of SF to suppress the SSS-III-specific antibody response. However, adsorption of SF with Ig+ (B) cells from mice immunized with 0.5 microgram SSS-III completely removed the suppressive activity. Significant (p less than 0.05) suppression of the antibody response was observed only when SF was administered (i.v.) 24 hr before to 24 hr after immunization with 0.5 microgram of SSS-III. These results suggest that suppressor T cells generated in response to SSS-III function by releasing a soluble factor(s) that binds to determinants on B cells rather than antigen; this soluble factor(s) acts directly on antigen-stimulated B cells or inhibits the induction of amplifier T cells.     

Regulation of herpes simplex virus-specific cell-mediated immunity by a specific suppressor factor.

Our study was designed to investigate the nature of an antigen-specific suppressor factor generated by antigen-stimulated herpes simplex virus (HSV)-immune splenocytes. Factor SF-200, a 90,000- to 100,000-dalton fraction obtained after Sephacryl gel filtration, suppressed the generation of HSV-specific cytotoxic T-lymphocyte and lymphoproliferative responses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of SF-200 indicated that it contained an I-J+, anti-idiotypic protein. It was possible to adsorb the suppressor activity of SF-200 to an anti-I-J immunoaffinity column. The suppressor activity could be eluted from the immunoaffinity column with a low-pH buffer. The acid-eluted material was determined to be both I-J+ and reactive with anti-HSV antiserum by Western blot analysis. Both SF-200 and the I-J+ suppressor activity suppressed only HSV-specific cell-mediated immunity responses. However, it was possible to generate nonspecific suppressor activity by incubating the I-J+ suppressor factor with Lyt 1+ splenocytes from HSV-immune mice. The implication of these results with respect to the model for a suppressor cell circuit regulating HSV-specific cell-mediated immunity responses is discussed.     

Protection of mice from herpes simplex virus-induced retinitis by in vitro-activated immune cells.

A form of acute retinal necrosis occurred in the contralateral eyes of susceptible mice 1 week after each received a uniocular injection of live herpes simplex virus type 1 (HSV-1) in the anterior chamber. Although these mice did not develop systemic delayed hypersensitivity to virus antigens, their sera contained virus-specific antibodies at the time contralateral retinitis occurred. These findings suggest that systemic immunity might not be able to protect against contralateral retinitis. To explore this possibility further, we examined lymph nodes and spleens of intraocularly infected mice to determine whether their lymphoid tissues contained primed HSV-1-specific cytotoxic T cells. Virus-specific cytotoxic T cells were readily identified in these mice. We wondered why successful immune priming did not confer protection against HSV-1 retinitis. We examined this issue by evaluating the capacity of in vitro-generated, HSV-1-specific effector T cells to prevent retinitis by infusing these cells by various routes and at various times into mice that received an intracameral injection of HSV-1. The results revealed that virus-specific effector cells could prevent contralateral retinitis if injected intravenously or into the anterior chamber of the contralateral eye at the same time that virus was injected into one eye. However, the effector cells failed to prevent retinitis if they were injected into the same eye that received HSV-1 or if their intravenous administration was delayed until 24 h after the HSV-1 injection into the eye. We concluded that immune T cells can protect against contralateral retinal necrosis caused by uniocular injection of HSV-1 into the anterior chamber but only if they are administered during the first 24 h after virus infection. We propose that a retinitis-inducing process is set in motion during this early time interval postinfection. Once the process has been initiated and established, it is no longer susceptible to immune intervention. It would appear that mice that are susceptible to contralateral retinitis fail to mobilize a protective response quickly enough to ward off the establishment of the retinitis-inducing process and its disastrous eventuality.     

Protease-deficient herpes simplex virus protects mice from lethal herpesvirus infection.

Null mutants and attenuated mutants of herpes simplex virus (HSV) have been shown to induce immunity against challenge from wild-type virus. Null viruses with a defect in late gene products would be expected to express more viral genes than viruses with defects in essential early gene products and thus induce a better immune response. Herpesviruses encode a late gene product (serine protease) that is autocatalytic and cleaves the capsid assembly protein during viral replication. To determine whether a virus with a mutation in this gene could induce immunity, we constructed a recombinant virus containing the gusA reporter gene in the protease domain of the HSV type 1 UL26 open reading frame (ORF). Consistent with previous results (M. Gao, L. Matusick-Kumar, W. Hurlburt, S. F. DiTusa, W. W. Newcomb, J. C. Brown, P. J. McCann, I. Deckman, and R. J. Colonno, J. Virol. 68:3702-3712, 1994), recombinant virus could be isolated only from helper cell lines expressing the product of the UL26 ORF. Mice inoculated with the recombinant virus were unaffected by doses of virus that were lethal to mice infected with wild-type virus. Mice which were previously inoculated with the recombinant virus were also protected by a subsequent challenge with wild-type virus in a dose-dependent manner. These results indicate that recombinant viruses lacking the protease gene are avirulent but render protection from subsequent challenge.     

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.     

Different forms of membrane-associated herpes simplex virus glycoproteins induce functionally distinct subsets of herpes simplex virus-specific suppressor T cells.

Previous studies have shown that two types of virus-specific suppressor T cells (Ts) are induced in mice made tolerant with herpes simplex virus (HSV)-infected spleen cells (SC). One type of Ts blocks the afferent phase of the delayed hypersensitivity response to HSV (Ts-aff), and the other blocks the efferent or effector phase (Ts-eff). In this report we show that the induction requirements for these suppressor populations differ. Injection of SC infected for 6 h with HSV at a multiplicity of infection of 5 or less or treated with heat-inactivated virus induced only Ts-aff. Similar results were seen with SC incubated for 90 min in virus-free preparations containing only viral proteins. In contrast, the Ts-eff population was induced only by SC treated for 6 h with infectious HSV at a multiplicity of infection of 10. Collectively, these data indicate that Ts-aff are induced by adsorbed HSV antigens on SC, whereas Ts-eff are induced by nascent HSV antigens expressed on infected SC. In addition to their induction requirements, the two types of regulatory cells differ in their expression of effector function. Ts-eff but not Ts-aff require a cyclophosphamide-sensitive target cell in the immune recipient for suppressor function. The possible identity of this target cell and the significance of the different induction requirements between the two types of Ts are discussed.     

Role of suppressor T cells in herpes simplex virus-induced immune deviation.

Herpes simplex virus type 1 inoculated into the anterior chamber of the mouse eye induces suppression of anti-herpes simplex virus T-cell-mediated delayed hypersensitivity. This suppression is virus-specific, and mediated by splenic T lymphocytes, and it can be adoptively transferred to naive recipients.     

Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D.

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry. Truncated forms of gD lacking the transmembrane and cytoplasmic tail regions have been shown to bind to cells and block plaque formation. Using complementation analysis and a panel of gD mutants, we previously identified four regions of gD (regions I to IV) which are important for virus entry. Here, we used baculovirus vectors to overexpress truncated forms of wild-type gD from HSV type 1 (HSV-1) [gD-1(306t)] and HSV-2 [gD-2(306t)] and four mutants, gD-1(inverted delta 34t), gD-1(inverted delta 126t), gD-1(inverted delta 243t), and gD-1(delta 290-299t), each having a mutation in one of the four functional regions. We used an enzyme-linked immunosorbent assay and circular dichroism to analyze the structure of these proteins, and we used functional assays to study the role of gD in binding, penetration, and cell-to-cell spread. gD-1 and gD-2 are similar in antigenic structure and thermal stability but vary in secondary structure. Mutant proteins with insertions in region I or II were most altered in structure and stability, while mutants with insertions in region III or IV were less altered. gD-1(306t) and gD-2(306t) inhibited both plaque formation and cell-to-cell transmission of HSV-1. In spite of obvious structural differences, all of the mutant proteins bound to cells, confirming that binding is not the only function of gD. The region I mutant did not inhibit HSV plaque formation or cell-to-cell spread, suggesting that this region is necessary for the function of gD in these processes. Surprisingly, the other three mutant proteins functioned in all of the in vitro assays, indicating that the ability of gD to bind to cells and inhibit infection does not correlate with its ability to initiate infection as measured by the complementation assay. The region IV mutant, gD-1(delta 290-299t), had an unexpected enhanced inhibitory effect on HSV infection. Taken together, the results argue against a single functional domain in gD. It is likely that different gD structural elements are involved in successive steps of infection.     

Production of interferon by immune lymphocytes exposed to herpes simplex virus-antibody complexes.

Splenic leukocytes from rabbits immunized with herpes simplex virus (HSV) incorporated significant amounts of 3H-TdR and produced interferon after in vitro incubation with HSV antigens. In contrast, splenic leukocytes from nonimmune rabbits did not incorporate 3H-TdR or produce interferon when incubated with HSV antigens. Antigen-antibody complexes consisting of HSV antigens and anti-HSV antibody also were capable of stimulating immune leukocytes to incorporate 3H-TdR and to produce interferon.     

Synthesis of herpes simplex virus DNA in a soluble nuclear extract.

A soluble extract prepared from nuclei of HeLa cells infected with herpes simplex virus type 1 has been found to synthesize herpes DNA in a process comparable to that observed in intact nuclei. The incorporation of [³H]dTTP has an absolute requirement for Mg⁺⁺ and for the other three deoxyribonucleoside triphosphates, and is relatively independent of added ATP. The reaction product, although of relatively short chain length, bands in CsCl density gradients at the density of herpes DNA and is essentially free of labeled cell DNA. Incorporation of BrdUTP results in a density shift suggesting extensive replication of endogenous DNA sequences.     

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.     

Synthetic glycoprotein D-related peptides protect mice against herpes simplex virus challenge.

Glycoprotein D (gD) of herpes simplex virus (HSV) protects mice from a lethal challenge by either HSV type 1 (HSV-1; oral) or HSV-2 (genital). We evaluated whether synthetic peptides representing residues 1 through 23 of gD (mature protein) can be used as a potential synthetic herpesvirus vaccine. The immunogenicity of the peptides was demonstrated by the biological reactivity of antipeptide sera in immunoprecipitation and neutralization assays. All sera which immunoprecipitated gD had neutralizing against both HSV-1 and HSV-2. The highest titers were found in animals immunized with the longest peptides. The region of residues 1 through 23 was immunogenic regardless of whether the type 1 or type 2 sequence was presented to the animal. Immunization of mice with gD or synthetic peptides conferred solid protection against a footpad challenge with HSV-2. However, the peptides were not as effective as gD in protection against an intraperitoneal challenge. The results suggested that synthetic vaccines based on gD show promise and should be more rigorously tested in a variety of animal models.     

An analysis of factors influencing the isolation rate of herpes simplex virus.

Attempts were made to improve the rate of isolation of herpes simplex virus (HSV) from clinical specimens by minimizing loss of virus infectivity during transportation and employing the most sensitive cells for isolation. Basical analyses using standard strains of type 1 and type 2 HSV indicated that virus titer decrease was marked even at low temperatures in environments free of proteinous stabilizer such as normal serum or tissue extract, negating the generally held concept that HSV is stable in distilled water. YLE (Earle-lactalbumin HYDROLYSATE-YEAST EXTRACT) medium containing 20% inactivated calf serum was determined to be a transport medium of choice, because degradation of suspended virus during storage and freeze-thawing was negligible and loss of virus during Millipore filtration was minimal. Special coating of the membrane could also be obviated by the use of this solution. In a cell susceptibility test using clinical specimens, secondary rabbit kidney (SRK) cells were the most sensitive, showing a quick development of cytopathic effect. Vero and RK-13 cells were the second best, whereas monkey kidney, HeLa and L cells were far less sensitive. A total of 136 specimens from suspected cases, sent by dermatologists, were tested using SRK cells, and 99 strains of type 1 and 15 strains of type 2 HSV were isolated. Excluding one case from which vaccinia virus was isolated, the isolation rate of HSV was 84.4%.