Vaccine-induced immune responses in rodents and nonhuman primates by use of a humanized human immunodeficiency virus type 1 pol gene

Abstract

A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4+ and CD8+ T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol-based HIV-1 vaccine component.

FIG. 1.

Detection of in vitro pol expression from cell lysates of 293 cells transfected with V1Jns-pol_IIIB or V1R-opt-pol. Bands were detected using antiserum from an HIV-infected human subject. Lanes: 1, mock or no-vector control; 2 and 3, extracts from cells transformed with 1 μg of V1Jns-pol_IIIB or V1R-opt-pol, respectively; 4, blank; 5 and 6, extracts from cells transformed with 10 μg of V1Jns-pol_IIIB or V1R-opt-pol, respectively. MW, molecular weight (in thousands).

FIG. 2.

Groups of BALB/c mice were immunized with different combinations of three doses of V1R-opt-pol (20 μg/dose) and/or vacc-opt-pol (10⁷ PFU). (A) Frequencies of Pol-specific T cells in splenocytes of BALB/c mice at the end of the multidose regimen (3 weeks after dose 3). Spleens were collected from three representative mice per cohort and pooled. The numbers of IFN-γ-secreting SFC per 10⁶ splenocytes were determined in the absence (mock) or presence of pools of Pol sequence-derived 20-mer peptides (Pool 1 and 2). The arithmetic means of the counts from triplicate wells are reported, along with the standard errors of the mean. The analysis was repeated 9 weeks after dose 3 and yielded similar trends. (B) Mapping of T-cell responses to select Pol peptides. Pooled splenocytes from three mice from the DNA+DNA+vacc cohort were assayed for IFN-γ secretion against each of the 20-mer peptides. ELISPOT data were plotted as a function of the number of amino acids of the N-terminal residue of the 20-mer peptide. Arrows are indicated above each peptide that reproducibly stimulated cytokine secretion in succeeding T-cell depletion studies. A conservative count of 1,500 per 10⁶ cells was assigned to sample wells where the spots could not be quantified because of high spot density. A total of 84 peptides were tested; the aa 784 to 803 peptide is missing because of difficulties in synthesis. Reported are the means of triplicate well normalized to a 10⁶ cell input; the standard errors did not exceed 20% of the mean values.

FIG. 3.

(A) Frequencies of antigen-specific IFN-γ-secreting cells in unfractionated (whole) and CD4⁺- and CD8⁺-depleted splenocytes from BALB/c vaccinees. Each antigenic peptide was also tested with splenocytes from untreated (naïve) mice. (B) ⁵¹Cr release CTL killing by effector cells (from BALB/c vaccinees) of P815 cells pulsed with each of the CD8⁺ 20-mer peptides. Effector cells were prepared from splenocytes of 10 BALB/c mice immunized following DNA+DNA+vacc regimen (at 1 week after dose 3) and were restimulated with a mixture of aa 354 to 373, 464 to 483, 474 to 493, and 934 to 953 for 6 days in the presence of IL-2. E:T, effector/target ratio.

FIG. 4.

Cellular immune responses in BALB/c mice vaccinated intramuscularly with one or two doses of different amounts of either pol_IIIB or opt-pol plasmids. At 3 weeks after the second immunization, frequencies of IFN-γ-secreting splenocytes (shown with the standard errors) were determined for boosted and unboosted cohorts (using pools of five spleens per cohort) against mixtures of either CD4⁺ peptides (aa 174 to 193, 564 to 583, 684 to 703, 794 to 813, 884 to 903, and 924 to 943) or CD8⁺ peptides (aa 354 to 373, 464 to 483, and 934 to 953) at a final concentration of 4 μg/ml per peptide.

FIG. 5.

Frequencies of Pol-specific IFN-γ-secreting cells (as measured by the ELISPOT assay) in rhesus macaques vaccinated with 5 mg of V1Jns-tpa-opt-pol and V1R-opt-pol DNA vaccines at 0, 4, and 8 weeks. Numbers of SFC per 10⁶ PBMCs against pool 1 (cross-hatched portion of the bars) and pool 2 (open portion of the bars) are reported for each monkey at different assay times; mock responses to dimethyl sulfoxide (no peptide) ranged from 0 to 24 per 10⁶ PBMCs and were subtracted from these reported responses.

FIG. 6.

Bulk CTL killing activity of Pol-peptide-pulsed BLCLs by effector cells derived from rhesus PBMCs following a 2-week restimulation with vacc-tpa-opt-pol. PBMCs were collected 8 weeks after dose 3. As background control, BLCLs were pulsed only with the dimethyl sulfoxide solvent (no peptide). The same responders were observed in the CTL assay conducted 4 weeks earlier. E:T, effector/target ratio.