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. 2009 Nov;83(21):10963-74.
doi: 10.1128/JVI.01284-09. Epub 2009 Aug 12.

Lentiviral vector-based prime/boost vaccination against AIDS: pilot study shows protection against Simian immunodeficiency virus SIVmac251 challenge in macaques

Anne-Sophie Beignon  1 , Karine Mollier, Christelle Liard, Frédéric Coutant, Sandie Munier, Julie Rivière, Philippe Souque, Pierre Charneau
Affiliations

Lentiviral vector-based prime/boost vaccination against AIDS: pilot study shows protection against Simian immunodeficiency virus SIVmac251 challenge in macaques

Anne-Sophie Beignon et al. J Virol. 2009 Nov.

Abstract

AIDS vaccination has a pressing need for more potent vaccination vectors capable of eliciting strong, diversified, and long-lasting cellular immune responses against human immunodeficiency virus (HIV). Lentiviral vectors have demonstrated efficiency not only as gene delivery vehicles for gene therapy applications but also as vaccination tools. This is likely due to their ability to transduce nondividing cells, including dendritic cells, enabling sustained endogenous antigen presentation and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. We show in a first proof-of-concept pilot study that a prime/boost vaccination strategy using lentiviral vectors pseudotyped with a glycoprotein G from two non-cross-reactive vesicular stomatitis virus serotypes elicited robust and broad cellular immune responses against the vector-encoded antigen, simian immunodeficiency virus (SIV) GAG, in cynomolgus macaques. Vaccination conferred strong protection against a massive intrarectal challenge with SIVmac251, as evidenced both by the reduction of viremia at the peak of acute infection (a mean of over 2 log(10) fold reduction) and by the full preservation of the CD28(+) CD95(+) memory CD4(+) T cells during the acute phase, a strong correlate of protection against pathogenesis. Although vaccinees continued to display lower viremia than control macaques during the early chronic phase, these differences were not statistically significant by day 50 postchallenge. A not-optimized SIV GAG antigen was chosen to show the strong potential of the lentiviral vector system for vaccination. Given that a stronger protection can be anticipated from a modern HIV-1 antigen design, gene transfer vectors derived from HIV-1 appear as promising candidates for vaccination against HIV-1 infection.

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Figures

FIG. 1.
FIG. 1.
Structure of the lentiviral vector. A schematic representation of the vector plasmid DNA containing HIV-1 cis-active elements used to produce nonreplicative lentiviral vector particles encoding SIVmac239 GAG is shown. LTR, long terminal repeat; cPPT, central polypurine tract; CTS, central termination sequence; ieCMV, human cytomegalovirus immediate-early promoter; ψ, encapsidation signal; RRE, Rev responsive element; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element.
FIG. 2.
FIG. 2.
Subcutaneous injection of lentiviral vector did not result in systemic inflammation. The presence of IFN-α (A), IL-6 (B), and TNF-α (C) in the plasma shortly after subcutaneous injection was measured by ELISA (white, before injection; dots, 1 h after; strips, 6 h after; gray, 1 day after; black, 1 week after). The absence of either significant (IFN-α and TNF-α) or major (IL-6) increases in their level suggested that there was not systemic inflammation induced by the in vivo administration of lentiviral vector particles, even at a high dose (2.5 × 108 TU/animal). These data did not exclude a local inflammation likely triggered by intrinsic pathogen-associated molecular pattern (PAMP) (7, 20, 43).
FIG. 3.
FIG. 3.
A prime/boost lentiviral vector-based vaccination strategy induces robust cellular immunity. The longitudinal follow-up of the SIVmac239 GAG specific T-cell responses was performed at various time points postprime (light gray), postboost (medium gray), and postchallenge (dark gray) by IFN-γ ELISPOT assay after restimulation of whole PBMC with pools of overlapping peptides encompassing SIVmac239 GAG p55. The individual GAG-specific cumulative responses of all six vaccinated animals injected with TRIP-SIVmac239 GAG (low dose, animals 20022 and 20089; medium dose, animals 20293 and 20056; and high dose, animals 20195 and 20158) (A), two control animals immunized with an irrelevant antigen (TRIP-GFP) at a high p24 dose (animals 21544 and 20456) (B), and unvaccinated animals (animals 15661, 14184, 15885, and 14468) (C) are shown. Arrows indicate when the first injection (three doses), the second injection (identical dose), and the SIVmac251 challenge were performed. Aldrithiol AT-2 inactivated SIVmac239 was also used to restimulate GAG-specific CD4+ and CD8+ T cells 2 weeks postboost in a whole PBMC IFN-γ ELISPOT assay. Background after coculture with the control microvesicles was subtracted (D). When the frequency of specific T cells was high and spots overlapped, the number of IFN-γ SFC/million was underestimated to 1,400 before the background was subtracted. Indeed, the saturation curve of the ELISPOT reader was calculated by using serial dilutions of PBMC, and the maximum number of spots enumerated per well appeared to be 280 spots, corresponding to 1,400 spots/million PBMC when 200,000 cells were cultured. The symbol “+” was added at the top of the histogram when saturation was reached for at least one pool of peptides. At 2 weeks postchallenge, it was not possible to quantify the number of spots in the control medium without peptide wells and thus to calculate the cumulative response for animal 20022 (noted as “++”). nd, Not determined.
FIG. 4.
FIG. 4.
Injected animals develop humoral responses to VSV-G used to pseudotype the vector particles. The presence of neutralizing antibody against the envelope used for pseudotyping was measured with an in vitro transduction assay. P4 cells (HeLa derived) were cultured in the presence of lentiviral vectors encoding GFP-pseudotyped with VSV-G Indiana (A) or VSV-G New Jersey (B) preincubated with plasma diluted at 1:20 from immunized animals collected at various time points (black, before priming; gray, 2 weeks postprime; dots, 8 weeks postprime; and white, 2 weeks postboost). The transduction efficacy was assessed by flow cytometry. In the absence of plasma and at the dose of vector used, 61 and 23% of P4 cells were GFP+ after transduction with lentiviral vectors encoding GFP pseudotyped with VSV-G Indiana and New Jersey, respectively.
FIG. 5.
FIG. 5.
Vaccinated macaques have an improved control of viremia compared to unvaccinated and GFP control animals. Plasma viral loads were monitored for 5 months postchallenge, twice a week during the first 3 weeks, then once a week during the next 3 weeks and finally once a month. Viremia of unvaccinated (panel A in blue), GFP control (panel A in green), and vaccinated animals (panel B in red), as well as the geometric mean for the naive and GFP control group (in black) versus the vaccinated group (in gray) (A to C) are shown. The geometric mean of viral replication levels was lower in the vaccinated group at all time points tested (C). An average 2 log10 fold reduction of viremia was observed at the peak of acute infection (E). The geometric mean viremia of the vaccinated animals (in gray) was also compared to that of progressor animals (14184, 21544, and 20456) in orange and of slow progressor animals (15661, 15885, and 14468) in light blue (D). Post-acute-phase viremia levels were lower in vaccinated animals than in progressor animals. *, P < 0.05.
FIG. 6.
FIG. 6.
The total and CD28+ CD95+ memory CD4+ T-cell compartments are well preserved in vaccinated macaques. Changes in the numbers of CD28+ CD95+ memory CD4+ T cells in the peripheral blood were monitored for 5 months postchallenge. The percentages of baseline CD28+ CD95+ CD4+ T cells of unvaccinated (panel A in blue), GFP control (panel A in green), and vaccinated animals (panel B in red), as well as the mean for the naive and GFP control group (in black) versus the vaccinated group (in gray) (A to C), are shown. Vaccinated animals showed a full preservation of their CD28+ CD95+ CD4+ T-cell compartment during acute infection in contrast to naive and GFP control animals (C) and no gradual depletion in the chronic phase in contrast to progressor animals (14184, 21544, and 20456) in orange (D). *, P < 0.05. CD28+ CD95+ memory CD4+ T cells for all animals are compared at the peak of acute infection (E). The dynamics of total CD4+ T cells in vaccinees (in gray) also differ from those of control animals (in black) (F). *, P < 0.05.
FIG. 7.
FIG. 7.
Individual MHC haplotypes of the animals included in the cohort. MHC haplotypes (H1 to H7) were determined by microsatellite analysis. Intact and recombinant haplotypes are indicated for each chromosome of each animal and are color-coded. White boxes indicate variant microsatellite allele sizes relative to the expected haplotype (these rare variants generally differ by the addition or loss of a single repeat unit).
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