Steve Cai

Vaccination of mice with herpes simplex virus type 1 glycoprotein D DNA produces low levels of protection against lethal HSV-1 challenge

The herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene was inserted into vectors pSVL or pRc/CMV under control of the SV40 late promoter or the human cytomegalovirus major immediate-early promoter, respectively. Intramuscular injection of mice with these gD-containing plasmids appeared to induce low levels of serum anti-gD antibody, as judged by the appearance of low levels of anti-HSV-1-neutralizing antibody and anti-gD ELISA responses in the serum of gD-DNA-vaccinated mice. As previously reported in other virus systems, vaccination with vector DNA also induced ELISA and neutralizing antibody titers. However, these titers were lower than those induced by the gD-containing plasmids. The ELISA and neutralization titers induced by the vectors appeared to be non-specific rather than directed at specific HSV-1 proteins, since serum from mice vaccinated with plasmid-gD immunoprecipitated significant amounts of gD from extracts of HSV-1-infected cells, while serum from mice vaccinated with vectors was unable to immunoprecipitate gD or any other obvious HSV-1 proteins. Neither pSVL-gD nor pRc/CMV-gD induced detectable lymphocyte proliferative or CTL responses. Vaccination with pSVL-gD provided a significant (P = 0.04, Fishers exact test), but low level of protection against lethal challenge with HSV-1. Vaccination with pRc/CMV-gD also appeared to provide a low level of protection against challenge, that was statistically significance at the 10% level (P = 0.054, Fishers exact test). Reports from numerous laboratories (including ours) have shown that vaccination with recombinantly expressed gD can provide very high levels of protection against HSV-1 lethal challenge. Thus, the results reported here suggest that vaccination with HSV-1 gD-DNA is not yet a useful alternative to a gD subunit vaccine.

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 naïve 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.

Both CD4+ and CD8+ T cells are involved in protection against HSV-1 induced corneal scarring

AIM To determine the relative impact of CD4+ T cells and CD8+ T cells in protecting mice against ocular HSV-1 challenge. METHODS CD4+ T cell knockout mice (CD4−/− mice), CD8+ T cell knockout mice (CD8−/− mice), and mice depleted for CD4+ or CD8+ T cells by antibody (CD4+ depleted and CD8+ depleted mice), were examined for their ability to withstand HSV-1 ocular challenge. The parental mice for both knockout mice were C57BL/6J. RESULTS These results suggest that: (1) both CD4+ deficient mice (CD4−/− and CD4+ depleted mice) and CD8+ deficient mice (CD8−/−, and CD8+ depleted mice) developed significantly more corneal scarring than their C57BL/6J parental strain; (2) the duration of virus clearance from the eyes of the CD4+ deficient mice was 4 days longer than that of the CD8+ deficient mice; and (3) the severity of corneal scarring in the CD4+ deficient mice was approximately twice that of the CD8+ deficient mice. CONCLUSIONS It was reported here that: (1) CD4+ and CD8+ T cells were both involved in protection against lethal ocular HSV-1 infection; and (2) CD4+ and CD8+ T cells were both involved in protection against HSV-1 induced corneal scarring.

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 β2m°/° (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 β2m°/° (MHC‐I–) mice by all three vaccines. The neutralizing antibody titres were similar for all three vaccines, but were only ≈ 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 2×107 plaque‐forming units (PFU) of HSV‐1. The live virus vaccine and the 7gPs vaccine also protected 80% of the β2m°/° mice against the same lethal HSV‐1 challenge dose. In contrast, in Ab°/° mice, none of the vaccines provided significant protection against the same lethal challenge dose of HSV‐1. However, at a lower challenge dose of 2×106 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 β2m°/° 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 β2m°/° 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‐1 challenge.

The Role of Interleukin (IL)-2 and IL-4 in Herpes Simplex Virus Type 1 Ocular Replication and Eye Disease

To assess the relative effect of interleukin (IL)-2- and IL-4-dependent immune responses on herpes simplex virus (HSV)-1 infection, naive, vaccinated, and mock-vaccinated IL-20/0 and IL-40/0 knockout mice were challenged ocularly with HSV-1. Naive IL-20/0 mice were significantly more susceptible to lethal infection than IL-40/0 or parental BALB/c mice. Vaccinated, IL-20/0, IL-40/0, and BALB/c mice induced similar neutralizing antibody titers and were completely protected against HSV-1-induced death and corneal scarring. Vaccinated and mock-vaccinated IL-20/0 mice had significantly higher HSV-1 titers in their eyes than BALB/c mice, while vaccinated and mock-vaccinated IL-40/0 mice had significantly lower HSV-1 titers in their eyes than BALB/c mice. Recombinant (r) IL-2 treatment of the IL-20/0 mice significantly reduced ocular HSV-1 replications, but rIL-4 treatment of IL-40/0 mice significantly increased ocular HSV-1 replications. Th1 (IL-2) cytokine responses may help protect mice against ocular HSV-1 challenge and reduce ocular HSV-1 replication.

Perforin pathway is essential for protection of mice against lethal ocular HSV-1 challenge but not corneal scarring

Perforin (cytolysin; pore-forming protein) is expressed in both CD8(+) cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and is a major factor responsible for the cytolytic activities of these cells. Both CD8(+) T-cells and NK cells are important in eliminating cells infected with certain viruses. We examined the role of perforin in a mouse model of HSV-1 infection using perforin-deficient mice. Naïve perforin knockout (perforin(0/0)) mice were more susceptible to lethal HSV-1 ocular challenge (60% survival), than naïve parental C57BL/6 (100% survival). In contrast, both C57BL/6 and perforin(0/0) mice had similar levels of HSV-1 induced corneal scarring. Vaccination of perforin(0/0) mice induced a significantly higher HSV-1 neutralizing antibody titer than vaccination of C57BL/6 mice, and the mice were completely protected against lethal ocular challenge. These results suggest that in naïve mice ocularly challenged with HSV-1, the perforin pathway was involved in protection against death, but not in protection against corneal scarring.

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.

The US5 Open Reading Frame of Herpes simplex Virus Type 1 Does Encode a Glycoprotein (gJ)

Based on sequence analysis, the protein encoded by the US5 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N-glycosylation site and was given the designation of glycoprotein J (gJ). However, the US5 gene product has not been identified and the identity of gJ as a glycoprotein has not been confirmed. We have cloned and expressed the DNA encoding the complete sequence of the US5 ORF, using a baculovirus expression system. Western blotting, using polyclonal antibody raised against synthetic US5 peptides, revealed two major baculovirus-US5-expressed protein bands with apparent molecular weights of 16–17 and 10 kD. The recombinant US5 was found on the membrane of Spodoptera frugiperda cells and was susceptible to tunicamycin, endoglycosidase H, glycosidase F and partially resistant to endoglycosidase F. Vaccination of mice with baculovirus-expressed US5 did not induce a neutralizing antibody to HSV-1 or provide protection against lethal HSV-1 challenge. However, serum from these vaccinated mice was able to recognize US5 in purified HSV-1 virions by Western blot analyses and on the surface of HSV-1-infected cells by immunofluorescence. These findings establish that US5 does encode a glycoprotein and confirm the appropriateness of naming the US5 gene product gJ.

Involvement of CD8+ T-cells in exacerbation of corneal scarring in mice.

Purpose. To determine the specific immune responses involved in the exacerbation of corneal scarring induced by HSV-1 in gK vaccinated mice. Materials and methods. BALB/c mice were vaccinated with HSV-1 glycoprotein K (gK) and ocularly challenged with HSV-1. Infiltration into the cornea of T cells and macrophages was monitored by immunocytochemistry, and the effect of depletion of CD4+ T-cells, CD8+ T-cells, or macrophages on corneal scarring was determined. Results. Following ocular challenge, CD4+ and CD8+ T-cells and macrophages were more abundant in the corneas of gK-vaccinated mice than in the corneas of mock vaccinated mice. Depletion of CD8+ T-cells, but not of CD4+ T-cells or macrophages, reduced the severity of corneal scarring in gK-vaccinated mice. Conclusions. We have shown that gK vaccination causes an overall increase in T cells and macrophages in the cornea after ocular HSV-1 challenge. The immunopathology induced by gK vaccination appears to be related to CD8+ T-cell activity, as depletion of these cells, but not other immune cells, reduced corneal scarring.

Corneal macrophage infiltrates following ocular herpes simplex virus type 1 challenge vary in BALB/c mice vaccinated with different vaccines

Macrophage cell infiltrates in the cornea were examined following ocular herpes simplex virus type 1 (HSV-1) challenge of vaccinated BALB/c mice. Mice were vaccinated with individual HSV-1 glycoproteins, cocktails of different HSV-1 glycoproteins, or live avirulent HSV-1 (strain KOS). Cryostat sections of cornea were taken at different times after challenge and reacted with M1/70, F4/80, BM8, or MOMA-1 monoclonal antibodies. The pattern of macrophage responses in the cornea differed depending on the vaccine that was given prior to HSV-1 ocular challenge. No macrophage response was detected in mice vaccinated with the highly protective 5gPs consisting of the five glycoproteins gB, gC, gD, gE, and gI. In contrast, mock vaccinated mice and mice vaccinated with gK, which is known to exacerbate HSV-1 induced eye disease, had high sustained macrophage responses. Mice vaccinated with 7gPs (5gPs+gG and gH) had moderate levels of macrophages. It appeared that (1) the most effective vaccines induced no detectable infiltrating macrophages in the eyes, while the least efficacious vaccines had very high levels of infiltrating macrophages; (2) presence of CD11b(+) cells in the cornea appeared to correlate with enhanced blepharitis, but did not appear to affect corneal scarring; and (3) presence of F4/80(+) cells in the cornea tended to correlate with increased corneal scarring.