HSV-infected Target Research Articles

Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity.

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-gamma and TNF-alpha-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV(+) individuals had significantly lower proportions of mature CD3(-)/CD161(+)/CD56(+) NK cells and of IFN-gamma-producing NK cells compared with noninfected donors, independent of CD4(+) T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3(-)/CD161(+)/CD56(+) NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-gamma. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-gamma, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-gamma secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.

Outcome of herpes simplex virus type 2 infection in guinea pigs.

Factors that influence the outcome of genital herpes simplex virus (HSV) infection were explored in a guinea pig model. The viral inoculum required to establish infection in 50% of animals (ID50) was similar for inbred (strain 2) and outbred (Hartley) guinea pigs. However, the viral inoculum required to produce clinical disease in 50% of the animals (CD50) was 10 times greater for strain 2 compared to Hartley animals. HSV infection of both inbred and outbred animals was more likely to result in death of weanling than adult animals. The duration and severity of genital disease and the magnitude of vaginal viral replication were similar for strain 2 and Hartley animals in both young and adult animals. The lethal dose for 50% of animals (LD50) was 100-fold greater than the CD50 for Hartley animals, but the LD50 and the CD50 were equal in strain 2 guinea pigs. Viral cultures of homogenized neural tissues from infected animals revealed that HSV ascended to the level of the temporal cortex in strain 2 guinea pigs while virus was never recovered above the lumbar spinal cord in Hartley animals. Endogenous peripheral blood mononuclear cell-mediated cytolytic activity against HSV-infected targets was greater prior to HSV inoculation in survivors compared to animals that died. A fatal outcome of genital HSV-2 may relate to the failure to limit CNS viral replication. Death is more common among guinea pigs that have low endogenous HSV-directed natural killer activity, such as occurs among strain 2 and young animals whether inbred or outbred.

Heteroconjugate antibodies enhance cell-mediated anti-herpes simplex virus immunity in vivo.

Heteroconjugate antibodies are generated by covalently linking two mAbs with different specificities. When anti-CD3 mAb, capable of activating effector T cells, is coupled to anti-herpes simplex virus (HSV) mAb, which binds HSV antigens on virally-infected target cells, the resulting heteroconjugate antibody can be used in vitro to enhance anti-HSV immunity. Specifically, these heteroconjugate antibodies can augment anti-HSV immunity among lymphocytes previously lacking cytotoxicity against HSV-infected target cells. However, the efficacy of these specialized reagents in enhancing anti-HSV immunity in vivo has not been determined. We report here that anti-HSV heteroconjugates used in an adoptive transfer murine model of HSV-1 infection inhibited HSV replication in vivo and improved long-term survival. These results demonstrate that heteroconjugate antibodies have a potential therapeutic role in enhancement of anti-HSV immunity.

Immune Modulation of Natural Killer Cell Cytotoxicity Against Herpes Infected Target Cells in Pregnancy

Natural killer cell cytotoxicity (NKC) is a nonspecific, primary immunodefense system active against a variety of pathogens, including herpes simplex virus (HSV). Evidence suggests that during pregnancy, NKC is attenuated. The regulatory mechanisms for this immune attenuation have yet to be defined. We examined two cytokines (interleukin-2 [IL-2] and alpha interferon [IFN]) for their ability to alter NKC responsiveness during pregnancy, utilizing an HSV-infected target cell model. Peripheral mononuclear effector cells were isolated from 19 pregnant and 19 nonpregnant subjects by Ficoll-Paque separation. These cells were incubated with IFN, IL-2, or media alone, and analyzed for %NKC by an 18 h chromium release assay. The percentage of NKC was lower using the effector cells from the pregnant subjects as compared to nonpregnant controls. Incubation with either IFN or IL-2 resulted in a significant augmentation of NKC in both the pregnant and nonpregnant derived cells. There were no differences in IL-2 dose requirements or levels of cytotoxicity achieved (43.1 +/- 6.8% vs. 44.4 +/- 6.8%, respectively) between pregnant and nonpregnant derived cells. The IFN-mediated augmentation of NKC was somewhat blunted in pregnancy both in terms of absolute levels of cytotoxicity achieved (26.1 +/- 3.9% vs. 37.2 +/- 4.9%, respectively) and dose response curves generated. These results demonstrate that NKC against HSV infected cells is attenuated during pregnancy and can be immunoregulated with the use of either IFN and IL-2. The restoration of NKC responsiveness with IFN, however, remains incomplete during pregnancy, suggesting that this cytokine's mechanism of action differs from that of IL-2.

Alternative mechanisms of natural killer cell activation during herpes simplex virus infection.

Although NK cells can kill both malignant cells and virus-infected cells without prior sensitization, it has remained unclear whether the mechanism by which an NK cell is activated in the presence of a tumor cell is similar to that induced by the presence of a virus-infected cell. In our experimental system using homogeneous populations of cloned human CD16+ NK cells, we found that HSV-infected target cells do not induce in the NK cells the same pharmacologically-active second messengers elicited by NK-sensitive tumor cells. Although phosphoinositide turnover and calcium signaling were generated in NK cells exposed to NK-sensitive tumor cells, the recognition of HSV-infected cells by NK cells did not result in similar transmembrane signaling. Furthermore, depending on the cell type infected by HSV, alternative mechanisms of cytotoxicity were employed. HSV-infected foreskin fibroblasts were rapidly and selectively killed by cloned NK cells without a requirement for IFN or accessory cells. In contrast to this direct cytotoxicity against HSV-infected foreskin fibroblasts, NK cell-mediated cytotoxicity against an HSV-infected fibrosarcoma cell line (1591) was dependent on IFN-alpha production by accessory cells. Importantly, in both systems of cytotoxicity, IFN-alpha activation of NK cells resulted in augmented killing against both infected and uninfected targets. These results suggest that NK cell activation induced during antiviral immunity is distinct from activation elicited during an antitumor response. These differences include the utilization of alternative forms of signal transduction and alternative mechanisms of cytotoxicity.

Herpes simplex virus-infected cells disarm killer lymphocytes.

Human endothelial cells or human foreskin fibroblasts infected with herpes simplex viruses (HSVs) potently inhibit the lytic activity of natural killer cells and interleukin 2-activated killer cells. The inhibition occurs after as little as 8 hr of viral infection and requires contact between effector cells and HSV-infected targets. Inhibition evidently stems from direct blockade of killer cell function because killer cells placed atop HSV-infected targets rapidly become incapable of lysing subsequently added HL-60 or K-562 cells. The impairment of killer cell function is prevented when protein glycosylation in HSV-infected cells is blocked with tunicamycin. These studies may be relevant for understanding the persistence of herpes simplex virus infections and, further, suggest a mechanism for failed immune surveillance.

Heteroconjugate antibodies enhance cell-mediated anti-herpes simplex virus immunity.

Impaired cell-mediated immunity predisposes individuals to severe systemic HSV infections. A potential approach for enhancing antiviral immunity is to alter the specificity of T cells and NK cells so that they become cytotoxic against HSV. We describe here the use of heteroconjugate antibodies to augment the killing of HSV-infected cells. Two different types of heteroconjugate antibodies were used: 1) CD3-specific mAb, covalently linked to HSV-specific mAb (e.g., anti-CD3 x anti-HSV-1 glycoprotein C); 2) FcR-specific mAb linked to HSV-specific mAb (e.g., anti-Fc gamma RIII x anti-HSV-1 glycoprotein D). Whereas freshly isolated, PBL were not cytotoxic against HSV-infected target cells in a 5-h 51Cr-release assay, co-incubation with either heteroconjugate resulted in significant cytotoxicity. In vitro activated PBL (anti-CD3 + IL-2) also became more potent killers of HSV-infected cells in the presence of each heteroconjugate. The specificity of anti-CD3 x anti-HSV-1 and anti-Fc gamma RIII x anti-HSV-1 gD for enhancing T cell and NK cell immunity, respectively, was confirmed by using cloned, homogeneous human T cell and NK cell lines as effectors. Kinetic analysis demonstrated that as soon as the infected cells began to express HSV glycoproteins on their surface they became susceptible to this enhanced killing. Prolonged culture of HSV-infected cells with heteroconjugate antibodies and effector cells also decreased the amount of viral replication that occurred, as measured in a plaque inhibition assay. These results suggest that heteroconjugate antibodies are potent immunotherapeutic tools that enhance anti-HSV immunity.

Natural killer cytotoxicity and antibody-dependent cell cytotoxicity to herpes simplex virus-infected target cells in murine pregnancy.

In vitro natural killer cytotoxicity (NKC) and antibody-dependent cell cytotoxicity (ADCC) activity against herpes simplex virus (HSV)-infected cells were evaluated in a pregnant murine model (C57Bl6 inbred strain). Virgin (n = 16) and pregnant (late gestation) mice (n = 15) were infected intraperitoneally with HSV, type 1. After 18 hr, a 0.5-ml aliquot of the peritoneal wash was frozen for virus plaque assay, and the cells were cultured in the 51chromium release assay for NKC and ADCC. %NKC (mean +/- S.E.) to HSV-infected targets was significantly suppressed (P less than 0.05) in pregnant mice, 10.3% +/- 1.9, compared to that of virgin mice, 32.5% +/- 2.5. This suppression was abrogated with HSV-specific antisera (%ADCC); 53.9% +/- 4.4 (pregnant) compared to 49.1% +/- 3.6 (virgin). The diminished NKC activity in pregnant mice was reflected in an increased mean number of virus particles in the peritoneal wash, 266 +/- 66 PFU/ml, compared to 38 +/- 11 PFU/ml in virgin mice (P less than 0.05). We concluded that NKC, but not ADCC, to HSV-infected targets was suppressed and that HSV elimination was impaired in pregnant mice.

Augmentation of human natural cytotoxic cell activity by leukotriene B4 mediated by enhanced effector-target cell binding and increased lytic efficiency.

The addition of leukotriene B4 (LTB4) to cytotoxicity assays measuring natural killer (NK) or natural cytotoxic (NC) cell activities resulted in significantly augmented killing of K562 or herpes simplex virus (HSV)-infected target cells, respectively. Since the mechanism of cytotoxicity implies several steps, including the binding of effectors to targets which is Mg2+-dependent and the programming of lysis of the target which is Ca2+-dependent, we undertook to define the step(s) at which LTB4 acted in augmenting cytotoxicity. Our results showed that LTB4 significantly increased the percentage of effector-target conjugates when K562- or HSV-infected targets were incubated with lymphocytes. Maximal binding occurred at a concentration of LTB4 of 1 X 10(-10) M. Preincubation of lymphocytes and not target cells with LTB4 was sufficient to observe the increased binding. PBML binding to and killing of the NK-resistant target clone I, derived from K562, was not enhanced by LTB4. In the absence of Ca2+, cytotoxicity was impaired and LTB4 could not restore it. Use of a single cell lytic assay demonstrated augmented efficiency of lysis of both K562 and HSV-infected targets in the presence of LTB4. These findings suggest that LTB4 may augment natural cytotoxicity by enhancing target cell recognition by cytotoxic effector cells and subsequently by augmenting their lytic efficiency.

Clonal analysis of T cell responses to herpes simplex virus: A HSV-specific, non cytolytic T cell clone displays specific helper activity for T cell response in vitro

A HSV-specific, Lyt 1+2- T cell clone (D7.1) was established by repetitive in vitro restimulation of in vivo HSV-1 primed draining lymph node cells with UV-inactivated homologous virus. Earlier work had shown that this clone could provide specific helper activity for HSV-1 primed B cells for antibody production in vivo. In this paper, further functional analysis has demonstrated that this clone and its subclones could also help unprimed T cells in the generation of effector DTH T cells in vitro. The helper activity was found to be antigen-specific and cell dose-dependent. In addition, this helper T cell clone showed little, if any, natural killer-like activity and was non-cytolytic towards both HSV-infected and uninfected syngeneic targets. However, they could exert potent cytotoxic activity towards P815 and EL-4 tumour targets in the presence of lectins.

HLA-restricted T lymphocyte-mediated cytotoxicity against herpes simplex virus-infected cells in humans.

Cytotoxic T lymphocytes (CTL) against herpes simplex virus (HSV) were induced in vitro from human peripheral blood lymphocytes by stimulation with HSV antigen. CTL generated by HSV type 1 (HSV-1) antigen stimulation killed not only HSV-1-infected target cells but also HSV type 2 (HSV-2)-infected target cells, though at a lower level. This evidence suggests that CTL against HSV recognize the HSV type-specific and type-common determinants on HSV-infected target cells. These CTL were generated from high responders against HSV-1 antigen as measured by antigen-specific T lymphocyte proliferation in vitro, but not to such an efficient degree from low responders. The cytotoxic activities of CTL against the allogeneic HSV-infected target cells were high when at least one of the HLA-A or -B antigens was shared. However, the HLA-A and -B nonidentical target cells were not killed effectively. The data presented here suggest the possibility of HLA restriction of HSV-specific CTL in humans.

Murine cellular cytotoxicity to syngeneic and xenogeneic herpes simplex virus-infected cells.

Cellular cytotoxicity of C57BL/6 adult mice peritoneal cells to xenogeneic (Chang liver) and syngeneic (BL/6-WT3) herpes simplex virus (HSV)-infected cells was analyzed in a 6-h 51Cr release assay. There was no difference in antibody-dependent cellular cytotoxicity to either target. There was no natural killer cytotoxicity to targets with cells from uninfected mice except at very high effector cell ratios. HSV-infected (2 X 10(4) PFU intraperitoneally 1 day previously) mice mediated significantly higher antibody-dependent cellular cytotoxicity and required less antibody (10(-5) versus 10(-2) dilution), fewer cells, and less time to kill than cells from uninfected mice. HSV-infected mice mediated natural killer cytotoxicity but preferentially killed syngeneic HSV-infected cells. Stimulation of cytotoxicity was not virus specific since influenza-infected mice mediated similar levels of cytotoxicity to HSV-infected targets. There was no difference in morphology (95% macrophage) or in the percentage of FcR-positive cells, but infected mice had more peritoneal cells and generated higher levels of superoxide in response to opsonized zymosan or phorbolmyristate acetate. These data demonstrate nonspecific virus-stimulated metabolic and effector cell function which may enhance clearance of virus in an infected host.

Cellular cytotoxicity to herpes simplex virus-infected cells of leukocytes from patients with serious burns

Patients with serious burns occasionally are inflicted with unusually severe herpes simplex virus (HSV) infections. Purified monocyte—macrophages (MP), lymphocytes (L), and polymorphonuclear leukocytes (PMNL) from 11 patients with serious burns were studied for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and natural killer cytotoxicity (NKC) to HSV-infected target cells in an 18-hr chromium release assay. PMNL-ADCC of the burn patients (29.1 ± 4.1) was significantly higher ( P < 0.01) than control subjects (17.2 ± 2.6) in the acute phase immediately following the burn. The patients' MP-ADCC (12.3 ± 2.7) was significantly lower ( P < 0.01) than control subjects' MP-ADCC (32.8 ± 8.2) during the thrid week postburn. There were no significant differences in MP-NKC, L-NKC, and L-ADCC between patients and controls. High PMNL-ADCC was correlated with the high number of immature PMNL in the blood of patients during the acute phase following a serious burn. Low MP-ADCC was noted at the time when severe HSV infection tends to occur in these patients. This may help to explain the burn patient's susceptibility to unusually severe HSV illness.

Virus-infected colostral cell cytokine stimulation of human leukocyte natural killer cytotoxicity.

Natural killer cytotoxicity is an important antiviral defense mechanism. Human peripheral blood mononuclear cells cultured with herpes simplex virus (HSV)-infected cells produced a cytokine. This substance stimulated adult natural killer cytotoxicity from 53.0 +/- 10.5% to 79.8% (P less than 0.01) against HSV-infected target cells. These data resulted in a calculated cytokine-dependent cellular cytotoxicity (CDCC) value of 65.8%. Cytokine production was not stimulated by uninfected cells and was independent of the presence or absence of antibodies to HSV in sera of donors and mononuclear cells. Cells from human colostrum also produced an HSV-stimulated cytokine which mediated CDCC by using both adult (19.8 +/- 3.9%) and neonatal (18.6 +/- 3.4%) mononuclear effectors cells. Colostral cell cytokine production was also independent of donor HSV serology. Not all colostral cultures produced the cytokine, and in general colostrum-stimulated CDCC was lower than peripheral blood leukocyte-stimulated CDCC. Colostral cell cytokine stimulation of neonatal natural killer cytotoxicity may account in part for the increased nonspecific resistance of breast-fed infants to viral infection.

937 HUMAN NEONATAL POLYMORPHONUCLEAR LEUKOCYTE (PMNL) ANTIBODY-DEPENDENT CELLULAR-CYTOTOXICITY (ADCC) TO HERPES SIMPLEX VIRUS (HSV) INFECTED CELLS

ADCC is an important antiviral immune mechanism. Human neonatal cells have lymphocyte ADCC and functional PMNL defects. We therefore analyzed the ability of dextran sedimented, ficoll hypaque centrifuged purified cord blood PMNL from full term healthy infants to kill HSV infected target cells in an 18 hr 51 Cr release assay. At an effector to target cell ratio (E:T) of 100:1, ADCC activity of PMNL from 10 cord blood samples (45.1±6.2) was no different than that of PMNL from healthy adults (46.8±9.6). E:T analysis from 100:1 to 30:1 revealed identical values for all 8 sets of neonatal and adult PMNL tested. Antibody concentration requirements were similar for all 3 sets of neonatal and adult cells tested with activity at immune sera concentrations of 1/20 but none at 1/1000. There was no difference in ADCC activity of PMNL from neonates born by vaginal (44.1 ± 11.3) or Caesarian section (45.8 ±7.8) deliveries.These data demonstrate the normal function of cord blood PMNL in ADCC and highlight the importance of antibody against HSV in neonatal HSV infection.Supported by NIH grants 1 PO1 HD 13021, and AI 14450

938 COLOSTRAL CELL (CC) LYMPHOKINE STIMULATION NEONATAL MONONUCLEAR CELL (MC) NATURAL KILLER CYTOTOXICITY (NKC) TO HERPES SIMPLEX VIRUS (HSV) INFECTED CELLS

NKC is an important antiviral defense mechanism. Neonates have low NKC to HSV infected cells. Human blood MC cultured for 18 hr. with HSV infected cells produced a lymphokine which stimulated adult MC-NKC from 53.0 ± 10.5% to 79.8 ± 12.8%, (p<.01)in an 18 hr. 51Cr release assay against HSV infected target cells. This resulted in a calculated lymphokine-dependent cellular-cytotoxicity (LDCC) of 65.8%. No active lymphokine was produced in the presence of uninfected cells. LDCC lymphokine production was independent of MC donor HSV serology. Cells from colostrum of most (8/10) women also produced an HSV stimulated lymphokine which mediated adult MC-LDCC (19.6 ± 4.2%) and was greater (p<.05) than matched MC lymphokine activity (6.7 ±2.6%) from these women. CC lymphokine production was also independent of donor HSV serology. Similarly, most (10/13) CC lymphokine cultures could stimulate neonatal MC-LDCC (17.4 ± 11.1%) as well as adult MC-LDCC. Colostral cell stimulation of neonatal NKC by LDCC may account for the increased non specific resistance of breast fed infants to viral infection. Supported by NIH grants 1 P01HD 13021 and Al 14450

Interferon induction of natural killer cytotoxicity in human neonates

Low natural killer cytotoxicity has been associated with serious herpes simplex virus infections. Neonates' mononuclear cells had significantly lower NKC to HSV infected (P less than 0.001) and uninfected (P less than 0.005) target cells than did adults' MC. Under appropriate conditions, both neonates' MC to NKC was increased by exogenous human leukocyte interferon. Although neonates' and adults' MC had a similar sensitivity to interferon, there was a significant (P = 0.05) lack of consistency in neonates' MC response. Twenty-three percent (4/17) of neonates' MC samples did not respond with increased NKC in the presence of interferon, unlike adults' MC, all of which demonstrated increased NKC. Neonates' MC did not suppress adults' MC-NKC. These data demonstrate the efficacy of interferon as an NKC stimulator in the neonate, but indicate a heterogeneous response of neonatal cells. We are encouraged with the prospect of clinical trials of interferon to treat severe viral infections in neonates. We urge the inclusion of immune surveillance in these trials.

Effect of herpes simplex virus infection on murine antibody-dependent cellular cytotoxicity and natural killer cytotoxicity.

Mice intraperitoneally inoculated with a sublethal dose of herpes simplex virus (HSV) produced immunoglobulin G antibody-dependent cellular cytotoxicity (ADCC) and radioimmunoassay (RIA) antibody as early as 3 days after infection. There was a rise in natural killer cytotoxicity (NKC) to infected and uninfected target cells 1 to 3 days postinfection mediated by nonadherent peritoneal cells (PC) in mice inoculated with HSV, but also with other substances commonly used in tissue culture media. HSV caused the highest and most consistent increase in NKC. PC-NKC, as ADCC, was inhibited by latex and silica, both macrophage inhibitors. PC-ADCC markedly declined 3 to 8 days after HSV inoculation. This was not due to a soluble or cellular suppressor factor, was not reversed by incubation or trypsin treatment of PC, was not associated with a change in PC Fc receptors, adherence, or acridine orange staining characteristics, and could not be induced by inactivated HSV. In vitro inoculation of PC with HSV similarly caused a reduction in the ability of PC to mediate ADCC to HSV-infected target cells. These data demonstrate the complex stimulatory and inhibitory interactions between virus and host defense mechanisms.

Herpes-Simplex-virus-specific, H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen.

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2k) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected taraget cells expressing the Dd alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2Dk determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for Dd alloantigen could be positively selected on H-2d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing Dd alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.

Specifically Immune Mause T-Cells Can Destroy H-2 Compatible Marine Target Cells Infected with Herpes Simplex Virus Types 1 or 2

Lymphocytes from the spleen (SL) or peritoneal cavities (PL) of HSV-1- or HSV-2-immunized C3H mice expressed pronounced cytotoxicity, as measured by 51Cr release assay, against L cells experimentally infected with HSV-types 1 or 2 but not against uninfected L cells. HSV-1 or HSV-2 immune lymphocytes induced substantially more 51Cr release when L-cell targets were infected with homotypic virus compared to those infected with heterotypic virus. Inasmuch as the cytotoxicity of specifically immune C3H SL for HSV-infected L cells was selectively eliminated by treatment with AKR anti-theta C3H serum plus complement, the effector cells in present system were theta-bearing T cells. Evidence has been provided which indicates that specifically immune T cells express cytotoxicity exclusively directed against different HSV-infected target cells (mouse embryo cells or mouse peritoneal macrophages) which share H-2 antigens with the effector cells.