Improved survival in rhesus macaques immunized with modified vaccinia virus Ankara recombinants expressing simian immunodeficiency virus envelope correlates with reduction in memory CD4+ T-cell loss and higher titers of neutralizing antibody

Abstract

Previous studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high viremia and AIDS following challenge with a pathogenic strain of SIV. Although all animals became infected, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines compared with animals that received nonrecombinant MVA. Most importantly, the reduction in viremia resulted in a significant increase in median and cumulative survival. Continued analysis of these animals over the subsequent 9 years has shown that they maintain a survival advantage, although all but two of the macaques have progressed to AIDS. Importantly, improved survival correlated with preservation of memory CD4(+) T cells in the peripheral blood. The greatest survival advantage was observed in macaques immunized with regimens containing SIV Env, and the titer of neutralizing antibodies to the challenge virus prior to or shortly following challenge correlated with preservation of CD4(+) T cells. These data are consistent with a role for neutralizing antibodies in nonsterilizing protection from high viremia and associated memory CD4(+) T-cell loss.

FIG. 1.

Plasma viral load (A) and peripheral blood CD4⁺ T-lymphocyte levels (B) in immunized and control macaques after challenge with uncloned SIVsmE660. (A) Sequential levels of plasma viral RNA are shown for animals immunized prior to challenge with nonrecombinant MVA as control or recombinant MVA expressing SIVsm gag-pol (MVA-GP), env (MVA-E), or both gag-pol and env precursors (MVA-GPE). Results are expressed as the number of SIV RNA copy equivalents per ml of plasma. Plasma samples having values under assay threshold sensitivity were given a value of 100 copy equivalents per ml. (B) Sequential CD4⁺ T-lymphocyte levels evaluated by fluorescence-activated cell sorting on whole-blood samples from the same animals as those in panel A.

FIG. 2.

Immunization with MVA-SIVsmH4 recombinants reduces viremia levels during acute and set point phases of pathogenic SIVsmE660 infection. (A and B) Viral RNA levels for individual macaques in the control group and each vaccinated group are presented, with bars indicating the geometric means of plasma viral loads with 95% confidence intervals for each group at peak (A) and set point (B) of viremia. The significance of differences between the control group and each individual vaccinated group was analyzed using an unpaired t test, and P values for these comparisons of vaccinated groups are indicated. (C) The survival of macaques challenged with SIVsmE660 significantly correlated with peak and set point (20 weeks). (D) Plasma viral RNA levels as shown by Spearman rank correlation test.

FIG. 3.

Immunization with MVA-SIVsmH4 recombinants is associated with preservation of central memory CD4⁺ T lymphocytes following SIVsmE660 challenge. (A) Flow cytometric gating strategy for identification of naïve (CD28^high CD95^low), central memory (CD28^high CD95^high), and effector memory (CD28^low CD95^high) CD4⁺ T cells is shown for a representative macaque from the control group (H387) and a long-term-surviving macaque with sustained control of viremia vaccinated with MVA-Env (H422). Profiles are shown for samples collected prior to challenge, 4 and 20 weeks post-SIV challenge, and time of death (H387) or 472 weeks postchallenge (H422). As indicated by the percentages of central and effector memory CD4⁺ T cells, progressive loss of these populations was observed in the control macaque. In contrast, these populations were preserved in the vaccinated macaque with sustained control of viremia. (B) Bar graphs depicting mean cell numbers of total (left), central memory (center), or effector memory (right) CD4⁺ T cells in PBMC samples of the various groups for 1 week (white) prechallenge and 4 weeks (black) and 20 weeks (gray) postchallenge show loss of central and effector memory cells in the control macaques and significantly better preservation of these cells in each of the three vaccinated groups. (C) Better preservation of mean central memory and effector memory CD4⁺ T cells in macaques showing sustained control (SC) of viremia compared to those with partial control (PC). (D) A significant inverse correlation of peak viremia was observed with the percentage of CD4⁺ T cells remaining at 4 weeks (P < 0.0383) and 20 weeks (P < 0.0162) postchallenge.

FIG. 4.

Improved survival rates for macaques vaccinated with MVA-SIV recombinants are associated with preservation of CD4⁺ central memory T cells. (A) Kaplan-Meier plot of cumulative survival indicates significant differences between the control group and groups immunized with MVA-SIV recombinants expressing SIV env. The comparison of survival in these groups of monkeys was done using a Gehan-Wilcoxon test. P values for groups of vaccinated macaques compared to the control group are indicated. (B) The absolute number of memory CD4⁺ T cells (left) or percentage of prechallenge memory CD4⁺ cells remaining (right) at 20 weeks postchallenge is shown for animals that survived less than 1 year, between 1 and 2 years, or more than 2 years. Significant preservation of memory CD4⁺ T cells was observed in animals that survived 1 to 2 years or longer. (C) Scatter plots of the correlation between the percentage of central memory CD4⁺ T cells left in peripheral blood at week 4 (left) or week 20 (right) post-SIVsm infection and survival (weeks) of control and vaccinated macaques. Monkeys immunized with MVA-SIV recombinants expressing SIV env (MVA-env or MVA-gag-pol-env) are shown by solid diamonds, and open diamonds indicate animals immunized with MVA-GP or control MVA. P values for the Spearman rank correlation test are indicated.

FIG. 5.

Prechallenge and early postchallenge levels of SIVsmE660-NAb in plasma of vaccinated macaques correlate with the degree of central memory CD4⁺ T-cell preservation in peripheral blood. (A) Titration curves of percent inhibition of virus infectivity by plasma samples of each macaque of the control group are shown for plasma samples collected prechallenge; at 4 days, 12 days, and 16 weeks postchallenge; and at necropsy. No neutralizing activity was detected until 16 weeks, with two animals (18655 and H426) showing very low titers of neutralizing activity consistent with rapid disease progression. (B) Bar graphs of reciprocal titers of NAb (log₁₀) to the challenge virus SIVsmE660 prechallenge; at day 4, day 12, and week 16 postchallenge; and at the time of necropsy in each vaccinated group. Titers of NAb are presented for each of the six monkeys per immunization group. Data are presented as the ID₅₀ neutralization titers determined by nonlinear regression of obtained virus infectivity inhibition curve as described in Materials and Methods. The assay limit of detection, which was the lowest reciprocal plasma dilution tested, was an ID₅₀ titer of >20. (C) Scatter plots of the correlations between percentage of central memory CD4⁺ T cells remaining in peripheral blood at week 4 post-SIVsmE660 infection and log of SIVsmE660-NAb titers in plasma from macaques immunized with MVA-SIV recombinants expressing SIV env (MVA-env or MVA-gag-pol-env). Data for prechallenge and day 12 postchallenge plasma samples are shown. P values for the Spearman rank correlation test are indicated.