Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques

Abstract

Human T cell leukemia/T-lymphotropic virus type 1 (HTLV-1) infection occurs by cell-to-cell transmission and can induce fatal adult T cell leukemia. Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of 10 vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 10⁸ HTLV-1-producing cells. In contrast, the 2 vaccinated macaques without NAb induction and 10 unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8⁺ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

Keywords: HTLV-1; cell-to-cell transmission; macaque model; neutralizing antibody; vaccine.

Graphical abstract

Figure 1

HTLV-1-specific antibody responses after vaccination (A) Plasma anti-HTLV-1 antibodies detected by line immunoassay after vaccination in group VA (top) and group VB macaques (bottom). The scores for relative levels of plasma anti-Env (gp46 and gp21) antibodies were determined based on the band (line) intensity in the line immunoassay. Scoring was as follows: −, negative; ±, marginal. Scores of 1–4 are positive, with 1 being the lowest and 4 the highest. V indicates the time points of vaccination. wks post-vac, weeks post-vaccination. (B) Plasma anti-HTLV-1 antibody titers measured by particle agglutination assay after vaccination in group VA (top) and group VB macaques (bottom). The lower limit of detection is a 1:16 dilution. Geometric means of data obtained by assay in duplicate are shown. The arrowheads indicate the time point of vaccination. (C) Plasma anti-HTLV-1 NAb titers measured by syncytia inhibition assay at 4 weeks after the last vaccination in group VA (week 16 after vaccination; left) and group VB macaques (week 20 after vaccination; right). ATL-040 and Jurkat cells were cocultured in the presence or in the absence of serially diluted heat-inactivated plasma, and the number of syncytia was counted for evaluation of syncytia inhibition. Endpoint plasma dilution titers resulting in 50% or greater inhibition were determined. The lower limit of detection is a 1:5 dilution. The median endpoint titer in three independent experiments is shown.

Figure 2

HTLV-1 Env-specific T cell responses after vaccination Env-specific CD4⁺ (open box) and CD8⁺ (closed box) T cell frequencies in PBMCs at two weeks after the last vaccination in group VA (at week 14 after vaccination; left) and group VB macaques (at week 18 after vaccination; right). Specific T cell frequencies less than 0.02% were considered negative.

Figure 3

HTLV-1 proviral loads after HTLV-1 challenge (A) Changes in HTLV-1 tax proviral loads in PBMCs after HTLV-1 challenge in group N (n = 10; top), group VA (n = 5; bottom left), and group VB macaques (n = 5; bottom right). The lower limit of detection is 2 copies/10⁵ cells. (B) Comparison of proviral loads at week (wk) 1 (left), wk 4 (middle), and wk 20 (right) after challenge among groups N, VA, and VB. The line indicates the median proviral load in each group. Statistical comparisons were performed by Dunn’s multiple comparison test. Group VA showed significantly lower proviral loads than group N at weeks 1 and 4. Group VB showed significantly lower proviral loads than group N at weeks 1, 4, and 20.

Figure 4

HTLV-1-specific antibody responses after HTLV-1 challenge (A) Plasma anti-HTLV-1 antibodies detected by line immunoassay after HTLV-1 challenge. The scores for relative levels of plasma anti-HTLV-1 Gag (p19 and p24) and anti-Env (gp46 and gp21) antibodies were determined based on the band (line) intensity in the line immunoassay. Scoring was as follows: −, negative; ±, marginal. Scores of 1–4 are positive, with 1 being the lowest and 4 the highest. Macaque N7 exhibiting positive only for the p24-specific line at week 0 was considered HTLV-1 naive before ATL-040 inoculation according to the INNO-LIA instructions. (B) Plasma anti-HTLV-1 antibody titers measured by particle agglutination assay after HTLV-1 challenge in group N (left), group VA (middle), and group VB macaques (right). Geometric means of data obtained by assay in duplicate are shown.

Figure 5

HTLV-1 proviral loads after CD8⁺ cell depletion in protected macaques VA1, VA4, VB1, VB2, and VB3 Changes in proviral loads in PBMCs after anti-CD8 antibody administration. Changes in CD8⁺ T cell frequencies (%CD8) in PBMCs are also shown. The initial anti-CD8 antibody administration was at week 148 in macaque VA1, at week 126 in macaque VA4, and at week 44 in macaques VB1, VB2, and VB3, after challenge, respectively. The arrowheads indicate the time point of initial anti-CD8 antibody administration.

Figure 6

Anti-HTLV-1 antibodies after CD8⁺ cell depletion in protected macaques VA1, VA4, VB1, VB2, and VB3 (A) Changes in plasma anti-HTLV-1 antibodies detected by line immunoassay after CD8⁺ cell depletion. wks post-a-CD8, weeks after initial anti-CD8 antibody administration. The scores for relative levels of plasma anti-HTLV-1 Gag (p19 and p24) and anti-Env (gp46 and gp21) antibodies were determined based on the band (line) intensity in the line immunoassay. Scoring was as follows: −, negative; ±, marginal. Scores 1 through 4 are positive, with 1 being the lowest and 4 the highest. (B) Changes in plasma anti-HTLV-1 antibody titers measured by particle agglutination assay after CD8⁺ cell depletion. Geometric means of data obtained by assay in duplicate are shown. (C) Changes in plasma anti-HTLV-1 NAb titers measured by syncytia inhibition assay after HTLV-1 challenge. The median endpoint titer in three independent experiments is shown. The arrowheads indicate the time point of initial anti-CD8 antibody administration.