The role of natural killer cells and interferon in resistance to acute infection of mice with herpes simplex virus type 1

The relative roles of interferon (IFN) and natural killer (NK) cells in herpes simplex virus type 1 (HSV-1) infection of mice were examined. Adoptive transfer of adult mouse leukocytes into 4- to 6-day-old suckling mice protected the recipients from HSV-1 infection, as judged by viral titers in the spleen 2 days postinfection. Protection was mediated by several classes of leukocytes, including those depleted of NK cell activity by antibody to asialo GM1 and those depleted of macrophages by size separation. Mice receiving these leukocytes produced significantly higher levels of IFN 6 hr postinfection (early IFN) than did HSV-1-infected mice not receiving donor leukocytes. Antibody to IFN, under conditions that blocked early but not late IFN synthesis, greatly enhanced HSV-1 synthesis in mice receiving leukocytes and completely removed the protective effect mediated by leukocytes. High doses of anti-asialo GM1 blocked both NK cell activity and early IFN production and resulted in high titers of HSV-1. This effect on virus synthesis was not seen if mice were given antibody 1 day postinfection. Lower doses of anti-asialo GM1, which still depleted NK cell activity but had no effect on early IFN production, did not enhance HSV-1 synthesis. Depletion of NK cell activity with a low dose of antibody had no effect on the reduced HSV-1 synthesis resulting from prophylactic IFN treatment or on the enhanced HSV-1 synthesis resulting from antibody to IFN treatment. Thus, resistance to acute HSV-1 infection in mice correlates with early IFN production but not with NK cell activity, suggesting that NK cells are not major mediators of natural resistance in this model and that the antiviral effect of IFN is not mediated by NK cells.     

NK and NKT cell-independent contribution of interleukin-15 to innate protection against mucosal viral infection

Interleukin-15 (IL-15) is essential for the development, maturation, and function of NK and NKT cells, which are critical components of the innate immune defense against viral infections. We recently showed that mice lacking IL-15 and/or NK/NKT cells are significantly more susceptible to intravaginal (IVAG) herpes simplex virus type 2 (HSV-2) infection than control mice. For this study, we examined whether IL-15 has any direct antiviral activity, independent of NK/NKT cells, in innate protection against HSV-2 infection. A sensitive enzyme-linked immunosorbent assay for murine IL-15 was developed and used to show that IVAG HSV-2 infection induces IL-15 in vaginal washes. Using immunohistochemistry, we detected IL-15-positive cells in the submucosa and vaginal epithelium following IVAG HSV-2 infection. Local, but not systemic, delivery of murine recombinant IL-15 (mrIL-15) to the genital mucosae of IL-15(-/-) and RAG-2(-/-) gamma(c)(-/-) mice, which both lack NK and NKT cells, resulted in significant reductions in HSV-2 titers in genital washes and 60% survival following IVAG HSV-2 challenge. Furthermore, we showed that IL-15 is important for CpG oligodeoxynucleotide (ODN)-induced innate protection against genital HSV-2 infection. While 100% of CpG ODN-treated RAG2(-/-) gamma(c)(-/-) mice, which are capable of producing IL-15 but lack NK/NKT cells, survived an IVAG HSV-2 challenge, only 60% of CpG ODN-treated IL-15(-/-) mice survived, and all of these mice had similar vaginal viral titers to those in control mice by day 3 postchallenge. Lastly, a treatment of RAW264.7 cells with mrIL-15 induced the production of tumor necrosis factor alpha and beta interferon (IFN-beta), but not IFN-alpha, and significantly protected them against HSV-2 infection in vitro. The results of these studies indicate that IL-15 can act independently of NK/NKT cells in mediating the innate defense against viral infection.     

Neurokinin 1 receptor signaling affects the local innate immune defense against genital herpes virus infection

We show that genital infection with neurotropic HSV type 2 (HSV-2) induced a significant increase of the neuropeptide substance P (SP) within the genital tract of mice. SP was shown to weakly interfere with the HSV-2 replication. Furthermore, lack of SP signaling through the use of mice deficient in the SP receptor, neurokinin 1 receptor (NK1R), revealed an important role for SP in the innate defense against HSV-2. NK1R-deficient mice had significantly enhanced levels of HSV-2 in the genital tract and in the CNS following infection and a significantly accelerated disease progression, which was associated with an impaired NK cell activity locally in the vagina. Lack of NK1R signaling did, however, not impair the animals' ability to mount a protective immune response to HSV-2 following vaccination with an attenuated virus. Both NK1R+/+ and NK1R-/- mice developed strong HSV-2-specific Th1 T cell responses following vaccination. No genital viral replication was observed in either vaccinated NK1R-deficient or NK1R+/+ control animals following a genital HSV-2 challenge, and all of these animals survived without any symptoms of disease. In conclusion, the present results indicate that SP and NK1R signaling contributes to the innate resistance against HSV-2 infection in mice.     

Susceptibility of CCR5-deficient mice to genital herpes simplex virus type 2 is linked to NK cell mobilization

Following genital herpes simplex virus type 2 (HSV-2) exposure, NK cells and T cells are mobilized to sites of infection to control viral replication and spread. The present investigation sought to determine the role of the chemokine receptor CCR5 in this process. Mice deficient in CCR5 (CCR5-/-) displayed a significant reduction in cumulative survival following infection in comparison to wild-type, HSV-2-infected controls. Associated with decreased resistance to viral infection, CCR5-/- mice yielded significantly more virus and expressed higher levels of tumor necrosis factor alpha, CXCL1, CCL2, CCL3, and CCL5 in the vagina, spinal cord, and/or brain stem than did wild-type mice. Whereas there was no difference in absolute number of leukocytes (CD45high), CD4 T cells, or CD8 T cells residing in the draining lymph nodes, spleen, spinal cord, or brain stem comparing HSV-2-infected wild-type to CCR5-/- mice prior to or after infection, there were significantly more NK cells (NK1.1+ CD3-) residing in the brain stem and spleen of infected wild-type mice. Functionally, NK activity from cells isolated from the brain stem of HSV-2-infected wild-type mice was greater than that from HSV-2-infected CCR5-/- mice. In addition, antibody-mediated depletion of NK cells resulted in an increase in HSV-2 levels in the vaginal, spinal cord, and brain stem tissue of wild-type but not CCR5-/- mice. Collectively, the absence of CCR5 expression significantly impacts the ability of the host to control genital HSV-2 infection, inflammation, and spread associated with a specific reduction in NK cell expansion, infiltration, and activity in the nervous system.     

In vivo significance of NK cell on resistance against virus (HSV-1) infections in mice

Susceptibility to infection with herpes simplex virus type 1 (HSV-1) was examined in euthymic as well as athymic nude mice which were continuously depleted of natural killer (NK) cell activity by i.v. injection of anti-asialo GM1. In those NK cell activity-depleted mice, the mortality rate of infection with HSV-1 and the virus titers in the brain, liver, and spleen were notably higher than in the control mice. The enhanced susceptibility was demonstrated only in the mice receiving anti-asialo GM1 and HSV-1 simultaneously, but not in the mice in which NK cell deletion was postponed by injecting the antisera 5 days after the virus inoculation. Interferon (IFN) production of peritoneal exudate cells was also reduced in the anti-asialo GM1-injected mice. The decline of resistance against HSV-1 infection proved to be primarily due to deletion of NK cells, but not due to the inability to produce IFN, because repeated injections of IFN increased the NK cell activity and prolonged the life of HSV-1-infected mice with an intact NK cell activity. In the NK cell activity-depleted mice, however, neither the NK cell activity nor the life span was improved by the administration of IFN.     

IL-18, but not IL-12, regulates NK cell activity following intranasal herpes simplex virus type 1 infection

Infection of the respiratory tract with HSV type 1 (HSV-1) can have severe clinical complications, yet little is known of the immune mechanisms that control the replication and spread of HSV-1 in this site. The present study investigated the protective role of IL-12 and IL-18 in host defense against intranasal HSV-1 infection. Both IL-12 and IL-18 were detected in lung fluids following intranasal infection of C57BL/6 (B6) mice. IL-18-deficient (B6.IL-18(-/-)) mice were more susceptible to HSV-1 infection than wild-type B6 mice as evidenced by exacerbated weight loss and enhanced virus growth in the lung. IL-12-deficient (B6.IL-12(-/-)) mice behaved similarly to B6 controls. Enhanced susceptibility of B6.IL-18(-/-) mice to HSV-1 infection correlated with a profound impairment in the ability of NK cells recovered from the lungs to produce IFN-gamma or to mediate cytotoxic activity ex vivo. The weak cytotoxic capacity of NK cells from the lungs of B6.IL-18(-/-) mice correlated with reduced expression of the cytolytic effector molecule granzyme B. Moreover, depletion of NK cells from B6 or B6.IL-12(-/-) mice led to enhanced viral growth in lungs by day 3 postinfection; however, this treatment had no effect on viral titers in lungs of B6.IL-18(-/-) mice. Together these studies demonstrate that IL-18, but not IL-12, plays a key role in the rapid activation of NK cells and therefore in control of early HSV-1 replication in the lung.     

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK-432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)-1. A single intraperitoneal (i.p.) injection of more than 10 micrograms of OK-432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 micrograms), produced only a marginal effect. A single i.p. administration of OK-432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK-432, coinciding with the times when it induced a survival effect on HSV-infection. Treating OK-432-treated mice with a combination of an anti-macrophage agent, silica, and an anti-NK cell agent, anti-asialo GM1 serum, before infection diminished the antiviral effect of OK-432. The OK-432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK-432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.     

Genital HSV-2 infection induces short-term NK cell memory

NK cells are known as innate immune cells that lack immunological memory. Recently, it has been shown that NK cells remember encounters with chemical haptens that induce contact hypersensitivity and cytomegalovirus infection. Here, we show the existence of NK cell memory following HSV-2 infection. Stimulation with HSV-2 Ags led to higher IFNγ production in NK cells that were exposed 30 days previously to HSV-2, compared to NK cells from naïve mice. More importantly, this increased production of IFNγ in NK cells was independent of B- and T- lymphocytes and specific for the HSV-2 Ags. We also showed that previously exposed NK cells in a B- and T-lymphocyte free environment mediate protection against HSV-2 infection and they are necessary for the protection of mice against HSV-2 infection. Collectively, NK cells remember prior HSV-2 encounters independent of B- and T- lymphocytes leading to protection against HSV-2 mediated morbidity and mortality upon re-exposure.     

Passive immunization of mice with monoclonal antibodies to glycoprotein gB of herpes simplex virus

To investigate the protective ability of monoclonal antibodies (MCAs) to viral glycoprotein in herpes simplex virus (HSV) infection, athymic nude mice were inoculated intracutaneously with HSV type-1 (HSV-1) in the midflank. Three hours after inoculation, one group of mice was passively immunized with one of a series of MCAs to glycoprotein gB of HSV-1, and a control group of mice was given phosphate buffered saline alone. The control mice died within 16 days after infection, whereas the mice passively immunized with any of the MCA showed suppressed development of skin lesions. Three of six mice given MCA failed to develop any visible lesions and no HSV could be isolated from the lumbar dorsal root ganglia of these mice 60 days after the challenge. BALB/c mice were also protected from infection with HSV type 2 by passive immunization with MCA to HSV-1 gB.     

Interleukin-15 and natural killer and NKT cells play a critical role in innate protection against genital herpes simplex virus type 2 infection

Interleukin-15 (IL-15), natural killer (NK) cells, and NK T (NKT) cells, components of the innate immune system, are known to contribute to defense against pathogens, including viruses. Here we report that IL-15(-/-) (NK(-) and NKT(-/+)) mice and RAG-2(-/-)/gamma(c)(-/-) (NK(-) and NKT(-)) mice that lack all lymphoid cells were very susceptible to vaginal infection with a low dose of herpes simplex virus type 2 (HSV-2). IL-15(-/-) and RAG-2(-/-)/gamma(c)(-/-) mice were 100-fold more susceptible and RAG-2(-/-), CD-1(-/-) (NKT(-)), and gamma interferon (IFN-gamma)(-/-) mice were 10-fold more susceptible to vaginal HSV-2 infection than control C57BL/6 mice. NK and/or NKT cells were the early source of IFN-gamma in vaginal secretions following genital HSV-2 infection. This study demonstrates that IL-15 and NK-NKT cells are critical for innate protection against genital HSV-2.     

Plasmacytoid Dendritic Cells Contribute to Systemic but Not Local Antiviral Responses to HSV Infections

Plasmacytoid dendritic cells (pDC) produce type I interferons (IFN-I) and proinflammatory cytokines in response to viruses; however, their contribution to antiviral immunity in vivo is unclear. In this study, we investigated the impact of pDC depletion on local and systemic antiviral responses to herpes simplex virus (HSV) infections using CLEC4C-DTR transgenic mice. We found that pDC do not appear to influence viral burden or survival after vaginal HSV-2 infection, nor do they seem to contribute to virus-specific CD8 T cell responses following subcutaneous HSV-1 infection. In contrast, pDC were important for early IFN-I production, proinflammatory cytokine production, NK cell activation and CD8 T cell responses during systemic HSV-2 and HSV-1 infections. Our data also indicate that unlike pDC, TLR3-expressing cells are important for promoting antiviral responses to HSV-1 regardless of the route of virus administration.     

Intrinsic resistance to viral infection. Mouse macrophage restriction of herpes simplex virus replication

Macrophages isolated from mice resistant to acute (lethal) infection with a neurovirulent isolate of HSV-1 express intrinsic resistance to viral infection in vitro. Bone marrow (BM), spleen (S), peritoneal (P), and thioglycolate-stimulated peritoneal (Pthio) macrophages isolated from resistant C57BL/6 Cr (B6) mice consistently restrict HSV-1 macromolecular synthesis earlier in the viral replicative cycle than do macrophages isolated from the same tissue sources from more susceptible DBA/2Cr (D2) mice. B6-BM (BM macrophages from B6 mice) restrict HSV macromolecular synthesis at least at two points in the replicative cycle: 1) before the onset of alpha-protein synthesis and 2) between the onset of gamma 1 protein and DNA synthesis. D2-BM macrophages restrict HSV replication at about the time of DNA synthesis. B6-P macrophages restrict HSV replication shortly after gamma 1 protein synthesis, and D2-P macrophages inhibit the virus slightly later, but before DNA synthesis. B6-S macrophages restrict HSV replication at about the time of DNA synthesis, and D2-S macrophages inhibit replication after the onset of gamma 2 protein synthesis. Pthio macrophages are more permissive to HSV infection than BM, P, or S macrophages: restrictions in viral replication occur at the time of DNA synthesis in B6-Pthio macrophages, and after the onset of gamma 2 protein synthesis in D2-Pthio cells. These studies demonstrate that isolated macrophages from inbred mouse strains express intrinsic resistance to HSV infection that correlates with in vivo resistance to acute (lethal) infection. Intrinsic resistance to HSV-1 infection is due to restriction of viral macromolecular synthesis. HSV replication is inhibited in macrophages at multiple points in the viral growth cycle, depending on the tissue from which the cells are isolated.     

HF10, an attenuated herpes simplex virus (HSV) type 1 clone, lacks neuroinvasiveness and protects mice against lethal challenge with HSV types 1 and 2

Herpes simplex virus (HSV), a neurotropic virus, establishes life-long and, although rare, life-threatening infection in humans, and it may precipitate substantial medical and psychosocial morbidity. Here we show that HSV-1 strain HF clone 10 (HF10) exhibits impaired neuroinvasiveness in peripheral olfactory, vomeronasal and trigeminal conduits following intranasal as well as corneal inoculation. HF10 attenuation likely arises from multiple defects of HSV genes, so that HF10 will not revert to a virulent phenotype. Intranasal vaccination of mice with HF10 conferred significant protection against lethal challenge with HSV-1 and HSV-2 via the intranasal and intravaginal routes. Thus, we propose that HF10 explicitly meets the prerequisites for a candidate live attenuated HSV vaccine.     

Plasmodium chabaudi: Adoptive transfer of immunity with different spleen cell populations and development of protective activity in the serum of lethally irradiated recipient mice

Groups of lethally X-irradiated NIH mice were injected with either glass wool-filtered (g.w.) immune spleen cells or nylon wool enriched immune T cells from syngeneic mice immune to Plasmodium chabaudi, or g.w. normal spleen cells. After cell recipients were infected with P. chabaudi the three groups reached similar mean peak parasitaemias on Day 11. In passive transfer tests serum obtained from mice sacrificed at this time gave little protection compared to normal serum. On Day 14 g.w. immune spleen cell recipients had subpatent infections and enriched immune T-cell recipients had a lower mean parasitaemia than g.w. normal spleen cell recipients. Serum obtained on Day 14 from g.w. immune spleen cell recipients gave better protection after passive transfer than sera from enriched immune T-cell or g.w. normal spleen cell recipients. Day 14 serum from enriched immune T-cell recipients, but not from g.w. normal spleen cell recipients, produced some initial protection after passive transfer. These results suggest that the transferred immune spleen cells contributed to the observed humoral immunity in lethally irradiated recipient mice.     

Protection of mice against herpes simplex virus infection by a Lactobacillus casei preparation (LC 9018) in combination with inactivated viral antigen

Heat-killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV-1) in adult mice, but not significantly. The protection of mice against HSV-1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV-1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV-1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV-1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV-1 antigen may play an important role in host defense mechanisms against HSV-1 infection.     

Protection by polyI:polyC against infection with herpes simplex virus in mice pretreated with Corynebacterium parvum

The effect of Corynebacterium parvum on the protection by polyinosinic-polycytidylic acid (polyI:polyC) against lethal infection with Herpes simplex virus type 1 (HSV) was studied in mice. Pretreatment with C. parvum resulted in prolonged survival times in all experiments. One third of the mice survived an infection with 100 LD50, whereas all mice died when treated with polyI:polyC alone. Increased protection was observed up to 6 weeks after pretreatment and only seen when both C. parvum and polyI:polyC were given at the same site of injection (intraperitoneally). Protection against HSV correlated with increased interferon (IFN) activities induced by polyI:polyC in the peritoneal cavity of C. parvum-pretreated mice. In these mice, natural killer cell activity of peritoneal exudate cells (PEC) was also augmented in response to polyI:polyC. Protection was markedly decreased by intraperitoneal injection of silica or of an antiserum against murine IFN. It appears that increased local levels of IFN presumably produced by macrophages in response to polyI:polyC in C. parvum-pretreated mice play the major role in the antiviral defence in our model and that activation of NK cells may be a secondary effect of IFN.     

CD4+ T cell migration into the cornea is reduced in CXCL9 deficient but not CXCL10 deficient mice following herpes simplex virus type 1 infection

The role of CXCL9 and CXCL10 in the ocular immune response to herpes simplex virus type 1 (HSV-1) infection was investigated using mice deficient in either CXCL9 or CXCL10. CXCL10 but not CXCL9 deficient mice showed an increase in sensitivity to ocular virus infection as measured by an elevation in virus titer recovered in the tear film and corneal tissue. The increase in virus was associated with an increase in the expression of the chemokine CCL2 but no significant change in the infiltration of CD4(+) T cells or NK cells into the corneal stroma. In contrast, a significant reduction in CD4(+) T cell infiltration into the cornea was found in CXCL9 deficient mice following HSV-1 infection consistent with the absence of CXCL9 expression and reduction in expression of other chemokines including CCL3, CCL5, CXCL1, and CXCL10. Collectively, the results suggest a non-redundant role for CXCL9 and CXCL10 in response to ocular HSV-1 infection in terms of controlling virus replication and recruitment of CD4(+) T cells into the cornea.     

Induction of potent protection against acute and latent herpes simplex virus infection in mice vaccinated with dendritic cells

Background aimsDendritic cells (DCs) are the most potent antigen presenting cells of the immune system and have been under intense study with regard to their use in immunotherapy against cancer and infectious disease agents. In the present study, DCs were employed to assess their value in protection against live virus challenge in an experimental model using lethal and latent herpes simplex virus (HSV) infection in Balb/c mice.MethodsDCs obtained ex vivo in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 were loaded with HSV-1 proteins (DC/HSV-1 vaccine). Groups of mice were vaccinated twice, 7 days apart, via subcutaneous, intraperitoneal or intramuscular routes with DC/HSV-1 and with mock (DC without virus protein) and positive (alum adjuvanted HSV-1 proteins [HSV-1/ALH]) control vaccines. After measuring anti-HSV-1 antibody levels in blood samples, mice were given live HSV-1 intraperitoneally or via ear pinna to assess the protection level of the vaccines with respect to lethal or latent infection challenge.ResultsIntramuscular, but not subcutaneous or intraperitoneal, administration of DC/HSV-1 vaccine provided complete protection against lethal challenge and establishment of latent infection as assessed by death and virus recovery from the trigeminal ganglia. It was also shown that the immunity was not associated with antibody production because DC/HSV-1 vaccine, as opposed to HSV-1/ALH vaccine, produced very little, if any, HSV-1-specific antibody.ConclusionsOverall, our results may have some impact on the design of vaccines against genital HSV as well as chronic viral infections such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus.     

Ccr5 deficiency regulates the proliferation and trafficking of natural killer cells under physiological conditions

Chemokines were shown to govern the trafficking of immune cells and may also play important roles in the survival and activation of these cells. We report here that under physiological conditions, the bone marrow (BM), spleen, blood and liver of Ccr5, but not of Ccr1-deficient mice, contain reduced numbers of NK cells. NK cells in the BM of Ccr5-deficient mice proliferate to a lesser extent compared to WT mice. Furthermore, spleen NK cells derived from Ccr5-deficient mice that were transplanted into irradiated recipients failed to proliferate in the host. Ccr5, but not Ccr1-deficient NK cells, failed to migrate in vitro in response to RANTES and MIP-1β but not MIP-1β or SDF-1 and had reduced activation, lower expression levels of NK cell markers and a slightly reduced capacity to adhere to target cells and stimulate their killing. Using the polyI:C mouse model for NK trafficking, we found that in the absence of Ccr5, but not Ccr1, NK cells failed to accumulate in the liver. In contrast, using the influenza viral infection as a model to evaluate NK cell proliferation, we found that Ccr5-deficient NK cells in the BM had a higher proliferation rate than WT NK cells. These results suggest a role for Ccr5 in NK cell proliferation and circulation under physiological conditions and a complex role for Ccr5 in determining the fate of NK cells under pathological conditions.