Inclusion of CD80 in HSV targets the recombinant virus to PD-L1 on DCs and allows productive infection and robust immune responses

Abstract

CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC.

Figure 1. Construction and structure of the HSV-CD80 recombinant virus.

(A) The top schematic diagram shows the HSV-1 McKrae genome in the prototypic orientation. TR_L and IR_L represent the terminal and internal (or inverted) long repeats, respectively, and TR_S and IR_S represent the terminal and internal (or inverted) short repeats, respectively. U_L and U_S represent the long and short unique regions, respectively. The solid rectangle represents the very stable 2 kb LAT. (B) dLAT2903 (parental virus for HSV-CD80) has a deletion from LAT nucleotides −161 to +1667 in both copies of LAT and makes no LAT RNA. (C) HSV-CD80 was constructed from dLAT2903 by homologous recombination between dLAT2903 DNA and a plasmid containing the complete LAT promoter and the entire structural CD80 gene (including its 3′ poly(A) signal) as described in Materials and Methods.

Figure 2. Replication of HSV-CD80 recombinant virus in RS cells.

Subconfluent RS cell monolayers in triplicate from two separate experiments were infected with 1/cell of HSV-CD80 or parental dLAT2903 virus as described in Materials and Methods. Total virus was harvested at the indicated times PI by two cycles of freeze-thawing. The amount of virus at each time point was determined by standard plaque assays on RS cells. Each point represents the mean + SEM (n = 6).

Figure 3. Replication of HSV-CD80 recombinant virus in BM-derived DCs.

Subconfluent monolayers of DCs isolated from C57BL/6, 129SVE, and BALB/c mice were infected with 10 PFU/cell of HSV-CD80 or parental virus as described in Materials and Methods. In some experiments DCs from C57BL/6 mice were infected with 1 PFU/cell of each virus, while DCs from 129SVE were infected with 10 PFU/cell of wt HSV-1 strain McKrae. Virus yield was determined at the indicated times PI by standard plaque assays. Panels: A) DCs from C57BL/6 mice were infected at 1 PFU/cell of HSV-CD80 or parental virus; B) DCs from C57BL/6 mice were infected at 10 PFU/cell of HSV-CD80 or parental virus; C) DCs from 129SVE mice were infected at 10 PFU/cell of HSV-CD80, parental, and wt McKrae viruses; and D) DCs from BALB/c mice were infected at 10 PFU/cell of HSV-CD80 or parental virus. Each point represents the mean ± SEM (n = 6) from two separate experiments.

Figure 4. Level of HSV-1 immediate early, early, and CD80 in HSV-CD80 infected DCs.

Subconfluent monolayers of DCs from C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80 or parental virus as in Fig. 1. For ICP-0 and TK measurements, cells were isolated 4, 8, and 12 hr PI, while for CD80 measurements cells were harvested 2, 4, 8, 12, 24, and 48 hr PI. Total RNA was isolated and TaqMan RT-PCR was performed using ICP0-, TK-, and CD80-specific primers as described in Materials and Methods. ICP0 and TK mRNA levels were normalized in comparison to each transcript at 0 hr PI, while CD80 mRNA level was normalized to the level of CD80 in mock infected DCs. GAPDH was used as internal control. Each point represents the mean ± SEM (n = 6) from two separate experiments. Panels: A) ICP-0; B) TK, and C) CD80.

Figure 5. Expression of CD80 by HSV-CD80 in infected DCs.

A) Detection of HSV-1 gC in infected DCs. Subconfluent monolayers of DCs isolated from C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c and anti-HSV-1 gC antibodies and FACS analysis was performed (see Materials and Methods). Experiments were repeated twice; B) FACS analyses of infected DCs for expression of HSV-1 gC and CD80. Monolayers of DCs were treated as above. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-HSV-1 gC, and anti-CD80 antibodies and FACS analysis was performed. CD11c⁺ cells were gated for expression of HSV-1 gC and CD80. Experiments were repeated twice; C) Immunostaining of HSV-CD80 infected DCs. DCs were infected with 1 or 10 PFU/cell of HSV-CD80 or parental virus. At 12 and 24 hr PI, cells were stained with anti-HSV-1 gC antibody and subjected to IHC as descried in Materials and Methods; and D) Quantification of photomicrographs from C. Different areas of 3 slides/virus/pfu/time point from IHC described above were imaged and the number of HSV-1 gC⁺ cells was counted. Each point represents the mean ± SEM of HSV-1 gC⁺ DCs from 12 images.

Figure 6. Colocalization of PD-L1 on DCs to CD80 expressed by HSV-CD80 and its effect on virus replication.

A) FACS analyses of infected WT DCs. Subconfluent monolayers of DCs isolated from WT C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-HSV-1 gC, and anti-PD-L1 antibodies and three-color FACS analysis was performed. CD11c⁺ gated cells were analyzed for expression of HSV-1 gC and PD-L1. Experiments were repeated twice; B) FACS analyses of DCs from knockout mice. DCs from C57BL/6 WT, C57BL/6-PD-L1^-/- and C57BL/6-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-CD80, and anti-PD-L1 antibodies and three-color FACS analysis was performed. CD11c⁺ gated cells were analyzed for expression of CD80 and PD-L1. Experiments were repeated twice; C) Immunostaining of DCs from knockout mice. DCs from WT BALB/c, BALB/c-PD-L1^-/- and BALB/c-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV-CD80 or parental virus. At 24 hr PI, cells were reacted with anti-HSV-1 gC, anti-CD80, and anti-PD-L1 antibodies. DAPI is shown as a nuclear counter-stain; D) Replication of HSV-CD80 in DCs isolated from knockout mice. Subconfluent monolayers of DCs isolated from WT BALB/c, BALB/c-PD-L1^-/- and BALB/c-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV- CD80 or parental virus as described in Materials and Methods. Virus yield was determined at the indicated times by standard plaque assays. Each point represents the mean ± SEM (n = 6) from two separate experiments; and E) Replication of HSV-CD80 in WT C57BL/6 DCs in presence of blocking antibody. Subconfluent monolayers of DCs were incubated with 10F.2H11 antibody, irrelevant antibody, or no antibody and infected with 1 PFU/cell of HSV-CD80 as described in Materials and Methods. Virus yield was determined at the indicated times by standard plaque assays. Each point represents the mean ± SEM (n = 6).

Figure 7. Coculture of HSV-CD80 infected DCs with naive WT T cells reduces CD3⁺PD-1⁺ expression.

DCs from WT C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock-infected. At 24 hr PI, the infected DCs were incubated with T cells isolated from naive WT C57BL/6 mice at a 1:1 ratio. As a control some T cells were incubated without DCs. FACS analyses were carried out 24 post incubation using anti-CD3 and anti-PD-1 antibodies. Graphs show the contour of CD3⁺PD-1⁺ staining for each group. Panels represent HSV-CD80, Parental virus, Mock, and no DCs T cells. Number indicates the percent of PD-1⁺CD3⁺ expressing T cells per treatment. Experiments were repeated twice.

Figure 8. Co-culture of HSV-CD80 infected DCs with naive WT T cells increases IFN-γ expression.

DCs from WT C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock-infected as in figure 7. At 24 and 48 hr PI, the infected DCs were incubated with T cells isolated from naive WT C57BL/6 mice at a 1:1 ratio. As a control some T cells were incubated without DCs. At 24 and 48 hr PI, T cells were isolated and quantitative RT-PCR for IFN-γ expression was performed using total RNA. IFN-γ expression in control T cells that were not incubated with DCs was used to estimate the relative expression of IFN-γ transcript in T cells that were co-cultured with mock-infected or infected DCs. GAPDH expression was used to normalize the relative expression of IFN-γ transcript in co-cultured T cells. Each point represents the mean ± SEM from 3 experiments.