Envelope-modified single-cycle simian immunodeficiency virus selectively enhances antibody responses and partially protects against repeated, low-dose vaginal challenge

Abstract

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV(mac)239 or SIV(mac)251(UCD), neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV(mac)251(UCD), six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.

FIG. 1.

Animals were immunized with Env-modified single-cycle SIV and challenged by repeated, low-dose vaginal inoculation. A schematic representation of the wild-type SIV_mac239 Env appears at the top (A). Positions of potential sites of N-linked glycosylation (N-X-S/T motifs) are indicated by tree-like symbols. Features removed from each Env-modified strain are indicated (red). The M5 Env has asparagine-to-glutamine substitutions that eliminate the 5th, 6th, 8th, 12th, and 13th potential N-linked glycosylation sites in gp120 (B). The g123 Env has all three potential N-linked glycosylation sites in gp41 similarly eliminated (C). Variable loops 1 and 2 of the ΔV1V2 envelope are deleted (D). All of the modified envelopes have a glutamate-to-stop codon change at position 767 (E767*). Eight macaques received an intravenous injection consisting of a mixture of 5 μg p27 equivalents of scSIV_mac239M5, scSIV_mac239g123, and scSIV_mac239ΔV1V2 on weeks 0, 6, and 12 (E). All eight animals also received 15 μg p27 equivalents of VSV G trans-complemented scSIV_mac239 intravenously on weeks 18 and 24. Beginning on week 32, the animals were challenged vaginally with 1,000 TCID₅₀ (1 ng p27) of SIV_mac251_UCD twice per day on the same day each week for 20 weeks or until viral RNA was detected in plasma on two consecutive weeks.

FIG. 2.

Plasma viral RNA following each inoculation with single-cycle SIV. The level of viral RNA in plasma was measured independently for each of these strains using a quantitative, multiplex real-time RT-PCR assay for the unique sequence tags ggr, cao, and gsa cloned into each of the three scSIV strains (23). This assay has a threshold of detection of 30 copies of viral RNA per ml plasma (dotted line).

FIG. 3.

Plasmas neutralized Env-modified and lab-adapted SIV but not wild-type SIV. Neutralizing antibody titers were compared between the animals immunized with Env-modified scSIV that subsequently became infected (blue) or remained uninfected (green) and historical control plasmas from animals immunized with scSIV expressing wild-type Envs (black). Pooled preimmune plasma served as a negative control (gray). The dotted line indicates 50% inhibition of SEAP activity. Plasma samples collected 2 weeks after the third dose of scSIV were tested for neutralization (neut.) of SIV_mac251_LA (A), SIV_mac239M5 (B), SIV_mac239g123 (C), SIV_mac239ΔV1V2 (D), and SIV_mac239 (I). Fifty percent neutralization titers are shown for SIV_mac251_LA (E), SIV_mac239M5 (F), SIV_mac239g123 (G), and SIV_mac239ΔV1V2 (H). P values were determined by two-tailed Mann-Whitney U tests. Week 26 plasmas were tested for neutralization of SIV_mac239 (J). Plasma samples drawn on week 32 were tested for neutralization of SIV_mac251_UCD (K).

FIG. 4.

Virus-specific antibody responses were detected in mucosal secretions. Anti-gp120 IgG specific activity (SA) was monitored longitudinally in CVS of immunized animals prior to infection (black), from the week each became infected (blue), and for the two that did not become infected (green) (A). Mucosal antibody responses in the naïve animals were measured prior to infection (gray) and postinfection (red). The 20-week challenge phase is shaded. The dashed line indicates the limit of detection, defined as the mean plus 3 standard deviations of negative samples. Anti-gp120 IgG SAs in CVS and plasma are correlated (Spearman's r = 0.91; P < 0.0001) (B). The ratios of anti-gp120 SA in CVS versus plasma were significantly higher at the five prechallenge time points in the two animals that remained SIV negative (C) (ratio, 3.6; 95% CI, 3.1 to 4.0; P < 0.001). IgG samples containing readings below the limit of detection, defined as 3 standard deviations above the average SA for naïve controls, were excluded from correlative and ratio analyses (B and C). IgA SA was monitored against gp120 in CVS (D) and rectal secretions (E) and against viral lysate in CVS (F) and rectal secretions (G).

FIG. 5.

T-cell responses were detectable against all proteins expressed by scSIV. IFN-γ ELISPOT responses against peptide pools of overlapping 15-mers representing the Gag, Tat, Rev, Vif, Vpr, Vpx, Env, and Nef proteins were measured over the immunization period (weeks 0 to 32). The limit of detection was 50 spot-forming cells (SFCs) per 10⁶ PBMCs.

FIG. 6.

Virus-specific CD8⁺ T cells were detected in peripheral blood and the vaginal mucosa. CD8⁺ T cells binding the MHC class I tetramers Mamu-A*01 Gag_181-189 (CM9), Mamu-A*01 Tat_28-35 (SL8), Mamu-A*02 Nef_159-167 (YY9), and Mamu A*02 Gag_71-79 (GY9) were detected in peripheral blood (A to E). The limit of detection indicated by the dotted line is 0.05% of CD8⁺ T cells. The 20-week challenge period is shaded. Tetramer-positive CD8⁺ T-cell frequencies are shown prior to infection (black) and from the week each animal became infected (blue) (A, B, and E). Frequencies of tetramer-positive CD8⁺ T cells are shown for the two immunized animals that remained uninfected (green) (C and D). Similar frequencies of CD8⁺ T cells were tetramer positive among lymphocytes isolated from vaginal biopsies versus peripheral blood (F).

FIG. 7.

Animals were challenged by repeated, low-dose vaginal inoculation. The eight immunized animals (blue) and six naïve controls (red) were challenged vaginally with 1 ng p27 (1,000 TCID₅₀) of SIV_mac251_UCD for 20 weeks, beginning on week 32. Virus was administered twice on the days of challenge—once in the morning and again in the afternoon—on the same day each week. All six naïve control animals became infected by the 11th week of challenge, but two of the eight immunized animals remained uninfected after the 20th week.

FIG. 8.

Postchallenge plasma viral RNA loads. Viral loads were measured at weekly intervals beginning on week 32 using a real-time RT-PCR assay with a limit of detection of 30 copies of RNA per ml of plasma for the immunized animals that became infected (blue) or remained uninfected (green) and for the naïve controls (red) (A) (19). Viral load measurements were synchronized to the last week in which viral RNA was undetectable in each animal that became infected (B). Comparison of geometric mean viral loads for the infected animals revealed acute peak viremia was reduced by 0.72 log (P = 0.0038; two-tailed Mann-Whitney U test) and was lower by 1.1, 1.2, 1.1, and 1.0 log on weeks 4, 6, 8, and 12 in the immunized group (C). Set point viral loads were significantly reduced for the period between weeks 5 and 67 postinfection, centered on week 28, as determined by a mixed linear model analysis (P = 0.004; 0.96 log lower; 95% CI, 0.31 to 1.6). Due to progression to AIDS, naïve control animals Mm 230-04, Mm 215-98, Mm 358-01, and Mm 128-01 were euthanized weeks 16, 23, 43, and 51 postinfection, and immunized animals Mm 316-98 and Mm 158-02 were euthanized 38 and 44 weeks postinfection.

FIG. 9.

CD4⁺ T-cell populations postinfection. CD4⁺ T-cell counts were synchronized to the last week in which viral RNA was undetectable for each animal. The numbers of total CD3⁺ CD4⁺ (A), CD3⁺ CD4⁺ CD28⁺ CD95⁻ naïve (B), CD3⁺ CD4⁺ CD28⁺ CD95⁺ central memory (C), CD3⁺ CD4⁺ CD28⁻ CD95⁺ effector memory (D), and CD3⁺ CD4⁺ CCR5⁺ CD95⁺ memory (E) T cells per μl of whole blood were monitored.