Protection against simian immunodeficiency virus vaginal challenge by using Sabin poliovirus vectors

Abstract

Here we provide the first report of protection against a vaginal challenge with a highly virulent simian immunodeficiency virus (SIV) by using a vaccine vector. New poliovirus vectors based on Sabin 1 and 2 vaccine strain viruses were constructed, and these vectors were used to generate a series of new viruses containing SIV gag, pol, env, nef, and tat in overlapping fragments. Two cocktails of 20 transgenic polioviruses (SabRV1-SIV and SabRV2-SIV) were inoculated into seven cynomolgus macaques. All monkeys produced substantial anti-SIV serum and mucosal antibody responses. SIV-specific cytotoxic T-lymphocyte responses were detected in three of seven monkeys after vaccination. All 7 vaccinated macaques, as well as 12 control macaques, were challenged vaginally with pathogenic SIVmac251. Strikingly, four of the seven vaccinated animals exhibited substantial protection against the vaginal SIV challenge. All 12 control monkeys became SIV positive. In two of the seven SabRV-SIV-vaccinated monkeys we found no virological evidence of infection following challenge, indicating that these two monkeys were completely protected. Two additional SabRV-SIV-vaccinated monkeys exhibited a pronounced reduction in postacute viremia to <10(3) copies/ml, suggesting that the vaccine elicited an effective cellular immune response. Three of six control animals developed clinical AIDS by 48 weeks postchallenge. In contrast, all seven vaccinated monkeys remained healthy as judged by all clinical parameters. These results demonstrate the efficacy of SabRV as a potential human vaccine vector, and they show that the use of a vaccine vector cocktail expressing an array of defined antigenic sequences can be an effective vaccination strategy in an outbred population.

FIG. 1

(A) Recombinant Sabin poliovirus vector plasmid clones. Grey boxes indicate 2A proteolytic cleavage sites (GLTTY/GFGH). In both the pSabRV1 and pSabRV2 vectors, the first proteolytic-cleavage-site coding sequence is followed by a 5-glycine spacer (not marked) and, immediately prior to the second proteolytic cleavage site, the in-frame cloning sites (white boxes). In total, an additional 60 to 70 nt are added to the viral genome to create the vector. (B) Plaque assay of cloned Sabin 1 virus (pS1 derived) and the Sabin 1 recombinant virus vector (SabRV1) at 32°C. (C) Plaque assay of cloned Sabin 2 (pS2–10F derived) and the Sabin 2 recombinant virus vector (SabRV2) at 37°C. (D) SabRV1-SIV plaque assays. The titers of all SabRV1-SIV viruses made were determined by plaque assay. Growth of several representative viruses at 32°C is shown here. SabRV1 without an insert is shown as a control. (E) SabRV2-SIV plaque assays. The titers of all SabRV2-SIV virus constructs were determined by plaque assay. Growth of several representative viruses at 37°C is shown here. SabRV2 without an insert is shown as a control. (F) Library schematic. Map of SIV antigens used in SabRV1-SIV and SabRV2-SIV vaccine cocktails. Each of the numerically labeled fragments corresponds to the different SabRV-SIV constructs defined in Table 1.

FIG. 2

Propagation of vaccine viruses. (A) Stability of SabRV2 recombinant viruses passaged as a cocktail. Nine SabRV2-SIV viruses were mixed in equal amounts and passaged five times at an MOI of 0.1, for a total of at least 10 generations of viral replication. The P₁ virus is conservatively estimated as generation 2. Cocktail stocks were tested for the presence of the SIV inserts by RT-PCR using primers corresponding to the poliovirus sequence flanking the SIV inserts. The lane containing the SabRV2 empty-vector RT-PCR product is indicated by a V. The size of the SabRV2 band (427 bp) is the size of the virus containing no insert. Inserts were fully retained throughout all five passages. (B) Composition of SabRV2 cocktail over a series of passages. The passaged cocktail stocks were checked for the presence of the individual viruses by RT-PCR with primers specific for each SIV insert. Generation 1 indicates the original cocktail in which the nine P₀ viral stocks obtained directly from high-efficiency transfections were mixed in equal proportions. The generation 1 stock contained all nine viruses, as determined by RT-PCR. The middle and right panels show the presence of all nine SabRV2-SIV viruses both at generation 6 and at generation 10. The small bands present in Env15C and Env17 are minor deletion products representing less than 1% of the virus population (data not shown).

FIG. 3

Time line of vaccination and challenge. The numbers above the line indicate the weeks at which the various steps were performed.

FIG. 4

Serum anti-SIV antibodies. (A) Anti-SIV IgG titers in the sera of monkeys immunized with SabRV1-SIV and SabRV2-SIV. Seven cynomolgus macaques were inoculated intranasally with SabRV1-SIV at weeks 0 and 2 (indicated by ▵ below) and given intranasal boosters at weeks 19 and 21 with SabRV2-SIV (indicated by ▴ below). Monkeys are labeled as follows: 25231, ▪; 27244, ●; 27250, ▴; 27253, ♦; 27270, □; 27273, ○; and 28508, ▵. Titers indicated are reciprocal dilutions. A titer of 1 is stringently defined as an ELISA optical density reading 3 standard deviations above the average optical density for a group of negative-control monkeys (see Materials and Methods). (B) Anti-SIV IgA titers in the sera of SabRV1-SIV- and SabRV2-SIV-immunized monkeys. Symbols are as noted above. A clear anemnestic IgA response is evident for all seven macaques after SabRV2-SIV immunization at week 19. Symbols are as for panel A.

FIG. 5

Rectal anti-SIV antibodies. (A) Anti-SIV IgG titers in the rectal washes of monkeys immunized with SabRV1-SIV and SabRV2-SIV. Vaccinated monkeys were divided into two groups (upper and lower panels) for easier viewing of data. Titers indicated are reciprocal dilutions. A titer of 1 is stringently defined as an ELISA optical density reading 3 standard deviations above the average optical density for a group of negative-control monkeys (see Materials and Methods). (B) Anti-SIV IgA titers in the rectal washes of monkeys immunized with SabRV1-SIV and SabRV2-SIV. Vaccinated monkeys were divided into the same two groups (upper and lower panels) as in part A, for easier viewing of data. Monkeys are labeled as described in the legend to Fig. 4.

FIG. 6

Vaginal anti-SIV antibodies. (A) Anti-SIV IgG titers in the vaginal secretions of monkeys immunized with SabRV1-SIV and SabRV2-SIV. Vaccinated monkeys are divided into two groups (upper and lower panels) for easier viewing of data, as was done in Fig. 4 and 5. (B) Anti-SIV IgA titers in the vaginal secretions of monkeys immunized with SabRV1-SIV and SabRV2-SIV. Monkeys are labeled as described for Fig. 4.

FIG. 7

Western blotting. All seven monkeys had anti-SIV and anti-poliovirus antibody responses that were detectable by Western blotting. Each serum was immunoblotted against purified SIV virion-infected (left lanes) and poliovirus-infected (PV; right lanes) HeLa cell extracts. Positive controls used were SIV-positive rhesus serum (SIV⁺) and human poliovirus-immune serum (Polio⁺). Preimmune serum from monkey 27250 was used as a negative control (preimm.). SIV antigens recognized by each monkey are indicated by symbols on the left of the blot as follows: reverse transcriptase (RT), ▹; Gag, –; gp120, ●; and gp41, ○. Poliovirus VP1, recognized by all monkeys, is indicated by the leftward-pointing black triangles to the right of each blot. Bands do not necessarily line up precisely because several of the blots were done at different times, but SIV and poliovirus positive controls were always run as markers.

FIG. 8

SIV-specific CTLs. (A) SIV-specific CTLs after immunization with SabRV1-SIV. SIV-specific CTLs were detected using bulk PBMCs collected 2 weeks after immunization with SabRV1-SIV. Monkeys 25231, 27244, and 27250 tested positive for SIV Env-specific CTLs. □, negative-control target cells; ●, Env-expressing target cells. (B) SIV-specific CTLs after immunization with SabRV2-SIV. SIV-specific CTLs were detected using bulk PBMCs collected 6 weeks after immunization with SabRV2-SIV. Monkey 25231 tested positive for SIV Gag-specific CTLs and possibly Env-specific CTLs. □, negative-control target cells; ▴, Gag-expressing target cells; ●, Env-expressing target cells. E:T, effector/target ratio.

FIG. 8

SIV-specific CTLs. (A) SIV-specific CTLs after immunization with SabRV1-SIV. SIV-specific CTLs were detected using bulk PBMCs collected 2 weeks after immunization with SabRV1-SIV. Monkeys 25231, 27244, and 27250 tested positive for SIV Env-specific CTLs. □, negative-control target cells; ●, Env-expressing target cells. (B) SIV-specific CTLs after immunization with SabRV2-SIV. SIV-specific CTLs were detected using bulk PBMCs collected 6 weeks after immunization with SabRV2-SIV. Monkey 25231 tested positive for SIV Gag-specific CTLs and possibly Env-specific CTLs. □, negative-control target cells; ▴, Gag-expressing target cells; ●, Env-expressing target cells. E:T, effector/target ratio.

FIG. 9

Serum anti-SIV IgG antibody responses postchallenge. Antibody titers are shown for all animals postchallenge. Vaccinated monkeys (right panel) are indicated by the symbols used in previous figures: 25231, ▪; 27244, ●; 27250, ▴; 27253, ♦; 27270, □; 27273, ○; and 28508, ▵. Control monkeys (left panel) are indicated by symbols as follows: 26383, ★; 26385, ✖; 23414, ; 26405, ⋄; 26560, ▿; and 28118, ◂.

FIG. 10

SIV RNA loads. SIV RNA levels in plasma were measured postchallenge. All seven vaccinated monkeys and six control monkeys were challenged with an intravaginal inoculation of highly virulent SIVmac251 at week 30 of the experiment (challenge was at week 0). Vaccinated monkeys (right panel) are indicated by the symbols used in previous figures: 25231, ▪; 27244, ●; 27250, ▴; 27253, ♦; 27270, □; 27273, ○; and 28508, ▵. Control monkeys (left panel) are indicated by symbols as follows: 26383, ★; 26385, ✖; 23414, ; 26405, ⋄; 26560, ▿; and 28118, ◂. Vaccinated monkeys 27270 and 27244 were never positive for SIV RNA and appeared to be completely protected. The threshold sensitivity of the assay (indicated by a dashed line) is 100 RNA copy equivalents/ml. Data points below the threshold value are shown at 100. †, animal died between weeks 32 and 44 postchallenge.

FIG. 11

Clinical outcomes of vaginal challenge with SIVmac251. (A) Postchallenge CD4⁺ T-lymphocyte counts. CD3⁺ CD4⁺ T-lymphocyte counts in the peripheral blood of naive control cynomolgus macaques (left) and SabRV1-SIV- and SabRV2-SIV-vaccinated macaques (right) were determined on the day of challenge and through 36 weeks postchallenge. Symbols are as defined in the legend to Fig. 10. (B) Body weight. Weights of control macaques (left) and SabRV1-SIV- and SabRV2-SIV-vaccinated macaques (right) were measured on the day of challenge and through 44 weeks postchallenge. Weight is indicated as a percentage of the body weight measured on the day of challenge. Body weight changes of vaccinated animals were significantly better (P < 0.003) than those of the control monkeys. Symbols used are the same as for panel A. (C) Mortality curve. SabRV-SIV-vaccinated animals, ▪; control animals, ●.