Optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses

Abstract

One attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine is the use of viral vectors with a proven safety profile and an absence of preexisting immunity in humans, such as Newcastle disease virus (NDV). Several NDV vaccine vectors have been generated, and their immunogenicities have been investigated with different animal models. However, a systematic study to evaluate the optimal insertion site of the foreign antigens into NDV that results in enhanced immune responses specific to the antigen has not yet been conducted. In this article, we describe the ability of NDV expressing HIV Gag to generate a Gag-specific immune response in mice. We also have determined the optimal insertion site into the NDV genome by generating recombinant NDV-HIVGag viruses in which HIV gag was located at different transcriptional positions throughout the NDV viral genome. All recombinant viruses were viable, grew to similar titers in embryonated chicken eggs, and expressed Gag in a stable manner. Our in vivo experiments revealed that higher HIV Gag protein expression positively correlates with an enhanced CD8(+) T-cell-mediated immune response and protective immunity against challenge with vaccinia virus expressing HIV Gag. We also inserted a codon-optimized version of HIV gag in the described best location, between the P and M genes. Virus expressing the codon-optimized version of HIV gag induced a higher expression of the protein and an enhanced immune response against HIV Gag in mice. These results indicate that strategies directed toward increasing antigen expression by NDV result in enhanced immunogenicity and vaccine efficacy.

FIG. 1.

Scheme of the different recombinant NDVs expressing HIV Gag protein. Transcriptional units containing 1.5 kb HIV gag were inserted in different positions in the NDV genome. In every new gag transcriptional unit, nucleotides were added in the untranslated region to follow the rule of six. The representation of the different genes is not to scale.

FIG. 2.

Growth kinetics of NDVs in embryonated chicken eggs. Ten-day-old embryonated eggs were inoculated with 100 PFU of the indicated NDV viruses. Eggs were incubated for 96 h, and allantoic fluids were harvested at different time points (24, 48, 72, and 96 h postinfection). NDV titers in the allantoic fluids were determined by measuring indirect inmunofluorescence in Vero cells.

FIG. 3.

Recombinant NDV viruses stably express HIV Gag. NDV-HIVGag viruses were passaged five times in 10-day-old embryonated eggs. Allantoic fluids obtained after the fifth passage were used to infect Vero cells, and expression of Gag in NDV-infected cells was visualized by indirect immunofluorescence. Anti-HN monoclonal antibody detected the presence of NDV-infected cells (B to F; mock, A). An anti-p24 monoclonal antibody (71-31) detected the expression of HIV Gag (panels H to L; mock, G).

FIG. 4.

Expression levels of HIV Gag, driven by the different recombinant NDVs. (A) Lysates from Vero cells infected with the NDV-HIVGag and NDV-GFP viruses were subjected to SDS-PAGE and were transferred to nitrocellulose membranes. Membranes were immunoblotted using an anti-NDV rabbit serum (α-NDV) and an anti-p24 (α-p24) human monoclonal antibody (71-31), followed by incubation with an anti-rabbit IgG peroxidase-labeled antibody and an anti-human IgG peroxidase-labeled antibody, respectively. (B) Lysates from Vero cells infected with the NDV-HIVGag and NDV-GFP viruses at 12, 24, 36, or 42 h postinfection were separated by SDS-PAGE, and Western blotting was performed using anti-NDV rabbit serum and anti-p24 mouse monoclonal antibody (24-4), followed by incubation with an anti-rabbit IgG peroxidase-labeled antibody and an anti-mouse IgG peroxidase-labeled antibody, respectively. (C) Vero cells were infected at an MOI of 1 with the indicated NDV viruses. Media from the infected cells were harvested at the indicated time points, and virus titers were measured by indirect immunofluorescence in Vero cells.

FIG. 5.

Challenge with Vac-HIVGag in mice immunized with NDV-HIVGag viruses. (A) Groups of nine mice were immunized intranasally with 5 × 10⁵ PFU of each NDV-HIVGag virus. Control mice were inoculated with 5 × 10⁵ PFU of NDV-GFP or with PBS. Three weeks after, animals were boosted with 10⁶ PFU of the same virus. Three weeks after the boost, three mice per group were challenged with 10⁶ PFU of Vac/wt or Vac-HIVGag. The rest of the mice were left for 51 days, after which they were sacrificed and their spleens were removed for quantification of Gag-specific CD8 T cells. (B) Five days after the infection with vaccinia viruses, mice were sacrificed, lungs were homogenized in 1 ml of PBS, and vaccinia titers in CV-1 cells were determined. (C) Fifty-one days after the second immunization, CD8⁺ T cells were selected and incubated with HIV Gag-specific peptide-pulsed cells in an ELISPOT assay to determine the number of HIV Gag-specific IFN-γ-secreting cells.

FIG. 6.

Generation of recombinant NDV containing a mammalian codon-optimized version of HIV gag. (A) A recombinant virus expressing a synthetic codon-optimized version of HIV gag (NDV-HIVGag-opt) inserted between the P/V and M genes was rescued following the same protocol described for earlier experiments. (B) Lysates from Vero cells infected with NDV-GFP, NDV-HIVGag-XbaI, or NDV-HIVGag-opt at an MOI of 1 were separated in an SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were immunoblotted using an anti-NDV rabbit serum and an anti-p24 mouse monoclonal antibody, 24-4, followed by incubation with an anti-rabbit IgG peroxidase-labeled antibody and an anti-mouse IgG peroxidase-labeled antibody, respectively.

FIG. 7.

Challenge with Vac-HIVGag in mice immunized with NDV-HIVGag-opt virus. (A) Groups of nine mice were immunized intranasally with 5 × 10⁵ PFU of NDV-HIVGag-XbaI or NDV-HIVGag-opt virus. Control mice were inoculated with 5 × 10⁵ PFU of NDV-GFP or PBS. Three weeks afterward, animals were boosted with 10⁶ PFU of the same viruses. Three weeks after the boost, three mice per group were challenged with 10⁶ PFU of Vac/wt or Vac-HIVGag. The rest of the mice were left for 28 days, after which they were sacrificed and their spleens were removed for quantification of Gag-specific CD8 T cells. (B) Five days after the infection with vaccinia viruses, mice were sacrificed and vaccinia titers in lungs were determined in CV-1 cells. (C) Twenty-eight days after the second immunization, splenocytes were isolated and incubated with HIV Gag-specific peptide-pulsed cells. ELISPOT assay was performed to determine the number of HIV Gag-specific IFN-γ-secreting splenocytes.

FIG. 8.

In vivo cytotoxicity assay. Groups of three mice were immunized using 5 × 10⁵ PFU or 5 × 10⁶ PFU of NDV-GFP, NDV-HIVGag-XbaI, or NDV-HIVGag-opt. Eight days later, spleens from naive mice were harvested and were stained either with 0.2 μM or 2 μM CFSE and then cultured with HIV Gag CD8-specific peptides or without peptides, respectively. The two splenocyte populations were mixed equally and injected intravenously into previously immunized mice. Eighteen hours later, spleens from recipient mice were harvested and survival of each population of stained splenocytes was assessed by flow cytometry.

FIG. 9.

Induction of cellular immune responses in mice at different times postvaccination and postchallenge. (A) Mice were immunized intranasally with 5 × 10⁵ PFU of NDV-GFP, NDV-HIVGag-SacII, NDV-HIVGag-XbaI, or NDV-HIVGag-opt virus. Three weeks afterward, animals were boosted with 10⁶ PFU of the same virus. Three weeks after the boost, mice were challenged with 10⁶ PFU of Vac-HIVGag. (B) Spleens were harvested at 10 days after the first immnunization, 7, 14, and 21 days after the boost, and 5 days after challenge. CD8⁺ T cells were selected and incubated with HIVGag-specific peptide-pulsed cells in an ELISPOT assay to determine the number of HIV Gag-specific IFN-γ-secreting cells. (C) Before and 5 days after challenge, splenocytes were harvested, incubated with the HIV Gag peptide, and subjected to intracellular staining after incubation with anti-mouse Alexa 647-conjugated CD8 and anti-mouse phycoerythrin-conjugated IFN-γ. Samples were analyzed by flow cytometry.