The importance of MHC-I and MHC-II responses in vaccine efficacy against lethal herpes simplex virus type 1 challenge.

To investigate the importance of major histocompatability complex (MHC) class I- and MHC class II-dependent immune responses in herpes simplex virus-1 (HSV-1) vaccine efficacy, groups of beta 2% (MHC I-) and Ab% (MHC II-) mice were inoculated with various vaccines, and then challenged intraperitoneally with HSV-1. Following vaccination with either live avirulent HSV-1, expressed HSV-1 glycoprotein D (gD), or a mixture of seven expressed HSV-1 glycoproteins (7gPs), Ab% (MHC-II-) mice developed no enzyme-linked immunosorbent assay (ELISA) or neutralizing antibody titres. In contrast, significant ELISA and neutralizing antibody titres were induced in beta 2m% (MHC-I-) mice by all three vaccines. The neutralizing antibody titres were similar for all three vaccines, but were only approximately 1/4 to 1/3 of that developed in C57BL/6 (parental) mice vaccinated with the same antigens. All three vaccines protected 100% of the wild-type C57BL/6 mice against lethal challenge with 2 x 10(7) plaque-forming units (PFU) of HSV-1. The live virus vaccine and the 7gPs vaccine also protected 80% of the beta 2m% mice against the same lethal HSV-1 challenge dose. In contrast, in Abo/o mice, none of the vaccines provided significant protection against the same lethal challenge dose of HSV-1. However, at a lower challenge dose of 2 x 10(6) PFU, all three vaccines protected 70-80% of the vaccinated Ab% mice (compared to only 10% survival in mock vaccinated controls). Thus, vaccination provided some protection against lethal HSV-1 challenge in both beta 2m% and Ab% mice; however, the protection was less than that seen in the parental C57BL/6 mice. In addition, Ab% mice were less well protected by vaccination than were beta 2m% mice. Our results suggest that (1) both MHC-I and MHC-II are involved in vaccine efficacy against HSV-1 challenge; (2) both types of responses must be present for maximum vaccine efficacy: and (3) the MHC-II-dependent immune response appeared to be more important than the MHC-I-dependent immune response for vaccine efficacy against HSV-I challenge.     

Vaccination with different HSV-1 glycoproteins induces different patterns of ocular cytokine responses following HSV-1 challenge of vaccinated mice.

We previously reported that vaccination of BALB/c mice with different baculovirus expressed HSV-1 glycoproteins induced varying degrees of protection against HSV-1 ocular challenge, ranging from complete protection to no protection, to exacerbation of eye disease. To correlate specific local immune responses with protection and exacerbation of corneal scarring, we examined immune cell infiltrates in the cornea after ocular HSV-1 challenge of vaccinated mice. Mice were vaccinated with gD, which completely protects against corneal scarring, gG, which produces no protection against corneal scarring, or gK, which exacerbates corneal scarring. Cryostat sections of cornea were taken at different times after challenge and examined for infiltrating cells containing IL-2, IL-4, IFN-gamma, IL-6, or TNF-alpha. No corneal infiltrates were seen before challenge or 1 day after ocular challenge in any groups. By days 3-7, many cells containing IL-4 and IFN-gamma, but few cells containing IL-2, had infiltrated into the corneas of gG or mock vaccinated mice. At the same times, many cells containing IL-2, but few cells containing IL-4 or IFN-gamma, were seen in the corneas of gD vaccinated mice. In contrast, the corneas of mice vaccinated with gK contained large amounts of IL-2, IFN-gamma, and IL-4. Our results suggest that: (1) corneas from gD vaccinated mice had no corneal disease and developed a response highly biased toward IL-2 responses; (2) corneas from gG or mock vaccinated eyes had significant corneal disease and developed a mostly IL-4 and IFN-gamma cytokine response; and (3) corneas from gK vaccinated mice had exacerbated corneal disease and developed strong IL-2, IL-4 and IFN-gamma cytokine responses.     

CD8+-dependent CNS demyelination following ocular infection of mice with a recombinant HSV-1 expressing murine IL-2

Demyelinating diseases comprise a spectrum of immunopathologic syndromes in which myelin, the fatty covering of nerve cell fibers in the brain and spinal cord, is destroyed. In this study, we have shown for the first time that ocular infection of BALB/c mice with a recombinant herpes simplex virus type 1 (HSV-1) expressing IL-2 (HSV-IL-2) results in CNS demyelination as determined by light microscopy and EM. The demyelinated lesions involve periventricular white matter, brain stem, and spinal cord white matter. Demyelination was detected in the CNS of infected mice up to 75 days (the longest time point tested) post HSV-IL-2 infection. In contrast, mice infected with HSV-IFN-gamma or HSV-IL-4, which are identical to HSV-IL-2 but express IFN-gamma or IL-4 instead of IL-2, did not exhibit demyelination. Control mice infected with wild-type HSV-1 or parental virus also remained free of these symptoms. During early times (days 3-7), post-infection with HSV-IL-2 virus, a T(H)1 + T(H)2 pattern of cytokines was produced by lymphocytes of infected mice while mice infected with HSV-IFN-gamma or control viruses produced a T(H)1 pattern of cytokine. By day 21 post-infection, all infected groups exhibited a T(H)1 pattern of response. Immunohistochemistry and FACS analyses of infiltrates in the brains and spinal cords of HSV-IL-2-infected mice showed elevations in CD4+ and CD8+ T cells and macrophages. However, T cell depletion studies suggest that only central memory CD8+ T cells are directly involved in the demyelination process, with macrophages being involved through a bystander effect.     

Improved protection from primary ocular HSV-1 infection and establishment of latency using multigenic DNA vaccines

PURPOSE: To compare the effectiveness of immunization with "naked" DNA corresponding to the genes encoding five HSV-1 glycoproteins, gB, gC, gD, gE, and gI (5gP DNA), with immunization with the five glycoproteins (5gP protein). Also, to compare immunization of 5gP protein in Montanide ISA 720 (SEPPIC, Paris, France), an adjuvant recently approved for use in humans, with immunization of 5gP protein in Freund's adjuvant. METHODS: BALB/c mice were vaccinated with 5gP DNA or 5gP protein emulsified in ISA 720 or Freund's adjuvant. Neutralizing antibody titers were determined by plaque-reduction assays. IL-2, -4, and -12 and IFN-gamma levels were determined by ELISA after in vitro stimulation of spleen cells. After ocular challenge with 2 x 10(5) plaque-forming units [pfu] per eye of HSV-1 strain McKrae, virus replication in the eye, survival, blepharitis, corneal scarring, and latency were determined. RESULTS: Neutralizing antibody titers (approximately 1:800-1:1200), corneal scarring (trace) and survival (100%) were similar for all vaccine groups, including 5gP DNA. Compared with the other vaccine groups, the 5gP DNA group had less ocular virus replication, as judged both by maximum virus titer and time of viral clearance. ISA 720 appeared more effective than Freund's against ocular virus replication and eye disease. The 5gP DNA-vaccinated mice had less blepharitis and latency than any other group and had the highest levels of IL-12 and IFN-gamma. All vaccine groups had similar levels of IL-2. CONCLUSIONS: The 5gP DNA vaccine appeared to be more effective than the corresponding protein subunit vaccine, regardless of adjuvant. Emulsification of the 5gP protein in ISA 720 appeared to be more effective than emulsification in Freund's adjuvant.     

Antibody-dependent enhancement of HSV-1 infection by anti-gK sera

We previously reported that vaccination of BALB/c mice with the baculovirus expressed HSV-1 glycoprotein K (gK) or passive transfer of gK purified IgG to naive BALB/c mice causes severe exacerbation of HSV-1 induced corneal scarring following ocular challenge. In addition, a productive chronic infection, rather than a latent infection, is found in most trigeminal ganglia. These phenomena are accompanied by a very high T(H)1+T(H)2 response in the eye (Ghiasi, H., Cai, S., Nesburn, A.B., Wechsler, S.L., 1996. Vaccination with herpes simplex virus type 1 glycoprotein K impairs clearance of virus from the trigeminal ganglia resulting in chronic infection. Virology 224, 330-333; Ghiasi, H., Cai, S., Slanina, S., Nesburn, A. B., Wechsler, S.L., 1997. Nonneutralizing antibody against the glycoprotein K of herpes simplex virus type-1 exacerbates herpes simplex virus type-1-induced corneal scarring in various virus-mouse strain combinations. Invest. Ophthalmol. Vis. Sci. 38, 1213-1221; Ghiasi, H., Hofman, F.M., Cai, S., Perng, G.C., Nesburn, A.B., Wechsler, S.L., 1999. Vaccination with different HSV-1 glycoproteins induces different patterns of ocular cytokine responses following HSV-1 challenge of vaccinated mice. Vaccine 17, 2576-2582). In the studies reported here, we investigated the hypothesis that anti-gK serum produces antibody-dependent enhancement (ADE) of ocular HSV-1 infection. We found that gK vaccinated mice had significantly higher HSV-1 titers in their eyes than gD or mock-vaccinated mice and that anti-gK sera enhanced HSV-1 infection in the macrophage cell line U937. In addition, passive transfer of anti-gK sera to naive mice 24 h prior to ocular HSV-1 challenge also increased viral replication. These results were consistent with ADE of HSV-1 by sera to gK. This suggests that the severely exacerbated corneal disease seen following HSV-1 ocular challenge of gK vaccinated mice is a result of ADE. The ability of gK sera to cause harmful ADE may impact HSV-1 vaccine development.     

The role of natural killer cells in protection of mice against death and corneal scarring following ocular HSV-1 infection

C57BL/6 mice depleted of NK (natural killer) cells with anti-asialo-GM1 antibody were more susceptible to lethal HSV-1 ocular challenge (12% survival) than control C57BL/6 mice (100% survival), CD4+ depleted mice (100% survival), CD8+ depleted mice (80% survival), or macrophage depleted mice (85% survival). NK depletion also resulted in significantly higher levels of HSV-1 induced corneal scarring than was seen with any of the other groups. C57BL/6 mice depleted of NK cells with PK136 (anti-NK1.1 antibody which is more specific for NK cells than is anti-asialo-GM1 antibody) were also more susceptible to HSV-1 ocular challenge than T cell or macrophage depleted mice. Vaccination completely protected NK depleted mice against death and corneal scarring. In contrast to C57BL/6 mice, in BALB/c mice, NK depletion had no effect on survival or corneal scarring following ocular HSV-1 challenge. Experiments with IFN-gamma knockout mice (IFN-gamma(o/o) mice) suggested that IFN-gamma played a minor role in protection of na?ve mice against death following HSV-1 challenge. However, IFN-gamma did not appear to be an important factor in protection against HSV-1 induced eye disease. Thus, protection against HSV-1 induced corneal scarring in naive mice appeared to be due to a non-INF-gamma NK function. Our results therefore suggest that NK cells were very important in protecting naive C57BL/6 mice but not vaccinated C57BL/6 mice against corneal scarring and death following ocular HSV-1 challenge.     

Papulonodular mucinosis indicating systemic lupus erythematosus

We describe an 18‐year‐old girl with systemic lupus erythematosus (SLE) who had cutaneous papulonodular mucinosis (PNM) as the first sign of SLE. She presented with multiple flesh‐coloured papules on the face, abdomen and limbs. Histological examination of a biopsy taken from a papule showed diffuse deposition of mucin throughout the dermis, and direct immunofluorescence of lesional skin showed a dermoepidermal junction band composed of IgG, IgM and C3, consistent with PNM. Investigations showed that that the patient had leucopenia, positive antinuclear and anti‐double‐stranded DNA antibodies and lupus nephritis. PMN can be an unusual clinical presentation of SLE.