Immune responses to Ad5-gag vaccine.
Cohorts of three to six rhesus monkeys were immunized intramuscularly with escalating doses (107, 109, and 1011 viral particles) of Ad5-gag at weeks 0 and 24. The frequencies of HIV-1 Gag-specific T cells were determined by Elispot assay with a peptide pool that encompassed the entire Gag sequence as described in Materials and Methods (Fig. ). The magnitude of vaccine-induced T-cell responses was dependent on the dose of Ad5-gag, with the best responses observed at 1011 viral particles. These responses peaked at 4 weeks following the first immunization and declined gradually until the second immunization was given at week 24. Anamnestic responses were observed following the boost, with peak levels that were comparable to or exceeded those observed at week 4 (Fig. and ).
FIG. 1. Frequencies of Gag-specific IFN-γ-secreting cells from monkeys immunized with Ad5-gag. These values are expressed as the number of SFC per 106 PBMCs. Naïve monkeys (adenovirus type 5 neutralization (neut) titers < 10) were immunized (more ...)
FIG. 3. Time course of induction of Gag-specific T-cell responses from PBMCs of representative monkeys that received 1011 viral particles and 109 viral particles of Ad5-gag at week 0 and week 24 (A). Mock responses were subtracted to give the reported levels (more ...)
To address the effects of preimmunity to adenovirus on immunization with the Ad5-gag vector, two additional groups of monkeys were given one or three injections of 1010 viral particles of an adenovirus type 5 virus that did not encode the vaccine antigen prior to administration of 1011 viral particles of Ad5-gag. Serum anti-adenovirus type 5 neutralization titers resulting from the preexposure ranged from 30 to 90 and from 270 to 810 for the single and multiple exposures of the irrelevant adenovirus type 5 vector, respectively. These levels appear typical of the range of antiadenovirus neutralizing antibody titers observed in adult, North American humans (our unpublished results). The presence of existing vector-specific immunity in the animals resulted in a partial reduction but not total ablation of the Gag-specific cellular immune response following immunization with two doses of Ad5-gag. These responses were comparable to those observed with the 109 virus particle dose of Ad5-gag in animals with no existing immunity to the vector (Fig. ).
The T-cell responses observed against the Gag antigen in the experimental animals exhibited functional, cytotoxic activities in CTL assays. All vaccinees, including those with established adenovirus type 5-specific immunity prior to Ad5-gag immunization, exhibited moderate to strong cytotoxicity against autologous Gag peptide-pulsed BLCL after a single immunization (Fig. ). Each animal was tested for CTL responses on a monthly basis for approximately a year following the first immunization. Cytotoxic responses were notably maintained in each animal throughout this period (data not shown).
FIG. 2. CTL responses in monkeys immunized with Ad5-gag. Peptide-pulsed autologous BLCLs were used as target cells at the indicated effector-to-target cell (E:T) ratios, treated with PBMCs (collected at week 8) from Ad5-gag vaccinees restimulated for 2 weeks (more ...)
The stability of both T-cell and B-cell immune responses was observed throughout an extended period of time. Figure shows longitudinal profiles of anti-Gag Elispot and anti-p24 Gag antibody responses. The levels of antigen-specific T cells remained significant for as long as a year following the booster. Ad5-gag also elicited substantial levels of circulating p24-specific antibodies (Fig. ). Based on the elicited humoral immunity against Gag, the animals that had preestablished adenovirus type 5 neutralization titers of 30 to 90 appeared to respond to the 1011 viral particle dose, similar to adenovirus-naïve monkeys that received 109 viral particles of Ad5-gag. Anti-Gag antibody titers induced by 1011 viral particles of Ad5-gag in animals with preimmunization neutralization titers of 270 to 810 were comparable to those induced by a 107 viral particle dose in naïve animals.
The levels of Gag-specific CD4+ and CD8+ T cells can be determined by intracellular cytokine staining (ICS). Figure shows the staining of PBMCs from a different yet representative set of rhesus macaques immunized with 1011 viral particle doses of Ad5-gag at weeks 0, 4, and 24. In the absence of Gag antigen stimulation, very little IFN-γ production was observed (<0.03% of CD3+ T cells). Upon overnight stimulation with the Gag peptide pool, cytokine production was detected from both CD8+ and CD4+ T cells. Substantially more Gag-specific CD8+ T cells than CD4+ T cells were observed in each animal. These data also support the observation of pronounced cytotoxic activities of T cells derived from Ad5-gag vaccinees.
FIG. 4. Intracellular IFN-γ staining of PBMCs from Ad5-gag vaccinees following overnight incubation in medium alone (mock) or in medium plus the Gag peptide pool (Gag 20-aa). CD3+ lymphocytes are shown and were characterized for CD8+ staining (more ...) Comparison of Ad5-gag and MVA-gag.
We compared the immunogenicity of the adenovirus type 5 vector to that of a recombinant MVA expressing the identical HIV-1 gag
gene. MVA is considered a standard for the elicitation of cellular immune responses (2
). Cohorts of three naïve monkeys were immunized with three doses of recombinant MVA expressing gag
(at either 107
PFU) or Ad5-gag
viral particles). The MVA vector had previously been confirmed for in vitro Gag expression in chicken embryo fibroblast cells and for Gag-specific immunogenicity in BALB/c mice (data not shown). Immunization of macaques with MVA-gag
resulted in relatively weak antigen-specific T-cell responses; the levels did not exceed 150 spot-forming cells (SFC)/106
PBMCs after three doses (Fig. ) and were significantly less than those observed in the Ad5-gag
FIG. 5. Frequencies of Gag-specific IFN-γ-secreting cells from monkeys immunized with Ad5-gag and MVA-gag. Priming immunizations were administered at weeks 0 and 4, followed by a booster shot at week 24 with the same virus. Shown are the mock-corrected (more ...)
It should be noted that two of three macaques that received MVA-gag at the higher dose (109 PFU) showed increased spot counts in their mock reaction mixes (109 to 238 SFC/106 PBMCs) as early as 4 weeks after the first dose (data not shown). In contrast to adenovirus type 5-treated monkeys, effector cells from the MVA-gag vaccinees generated by PBMCs restimulation with either vaccinia virus-gag or Ad5-gag did not exhibit any detectable bulk killing of Gag peptide pool-pulsed autologous BLCL at any time point (data not shown). Only one of six MVA-gag vaccinees elicited any detectable Gag-specific antibody response (140 mMU/ml at 4 weeks post-dose 3 for monkey V215).
Immune responses following DNA-adjuvant priming and viral boosting.
A potential limitation in the use of common adenovirus serotype vectors in human subjects may be the negative effect of existing neutralizing immunity against the viral vehicle, as observed in the preexposed monkeys (Fig. ). Hence, we explored the possibility of priming the immune responses with HIV-1 gag delivered by an immunologically inert vehicle such as DNA. The primates were then given a booster immunization with a low dose of Ad5-gag (107 viral particles) selected to mimic the effect of existing adenovirus type 5-neutralizing immunity on a 1011 viral particle dose of Ad5-gag (Fig. and ).
Cohorts of three to six macaques were given priming immunizations of 5.0 mg of gag-expressing DNA plasmid either in saline or formulated with adjuvants at weeks 0, 4, and 8. The DNA vector in saline was found to be immunogenic in macaques, with maximal postpriming responses averaging 111 ± 32 SFC/106 PBMCs (Fig. ). Formulation of DNA with the CRL1005-based adjuvant resulted in much improved T-cell responses to HIV-1 Gag (322 ± 46 SFC/106 PBMCs), while the DNA-alum formulation elicited intermediate responses. The subsequent Ad5-gag booster immunization resulted in consistent increases in T-cell frequencies to levels higher than the postpriming peak values. The best responses following the boost were observed in the animals that were primed with the DNA-CRL1005 vaccine. The peak responses in these animals reached levels of between 1,000 and 1,500 SFC/106 PBMCs. These response levels were comparable to those observed with multiple high doses (1011 viral particles) of Ad5-gag in previously unexposed animals (Fig. ).
FIG. 6. Frequencies of Gag-specific IFN-γ-secreting cells from PBMCs of macaques immunized following various DNA prime-adenovirus type 5 boost regimens. (A) Macaques were given multiple doses of 5 mg of V1Jns-gag DNA intramuscularly in phosphate-buffered (more ...)
All six animals given nonadjuvanted DNA had maximum responses of ≈600 SFC/106 PBMCs following the booster immunization. Gag-specific antibody levels in all animals increased by 100-fold following boosting with Ad5-gag; the postboost levels in DNA-CRL1005- and DNA-alum-primed animals were also five- to eightfold elevated compared to those of DNA-primed animals (data not shown). The noted increases in immune responses with the DNA-adenovirus type 5 combination vaccine strongly suggest synergy between DNA and adenovirus type 5 modalities, given that this low adenovirus type 5 dose is capable of eliciting only weak responses when used alone in naïve monkeys (Fig. and ). To determine the maximum potency of the DNA prime-adenovirus type 5 boost regimen, animals primed with the DNA-CRL1005-based vaccine were boosted with 1011 viral particles of Ad5-gag. The responses exceeded 2,500 SFC/106 PBMCs in two of three animals.
The phenotypes of the vaccine-induced anti-Gag T lymphocytes after the priming or booster immunization can be monitored by ICS methods. Figure shows the results of such analyses for a representative cohort of macaques immunized with DNA-CRL1005 followed by the low-dose Ad5-gag booster. Analyses of PBMCs collected before the booster dose revealed that the ratios of antigen-specific CD4+/CD8+ T cells can vary, ranging from CD4+- to CD8+-biased population. However, after boosting with Ad5-gag, a pronounced CD8+-biased response was observed in all animals. Hence, DNA-CRL1005-adenovirus type 5 prime-boost immunization appeared to result in cellular immune responses that were comparable to those induced by multiple high doses of Ad5-gag alone on the basis of levels and CD4+/CD8+ distribution of antigen-specific T cells.
FIG. 7. Intracellular IFN-γ staining of PBMCs from a representative cohort of macaques immunized with three doses of V1jns-gag formulated with CRL1005-based adjuvant (weeks 0, 4, and 8) followed by 107 viral particles of Ad5-gag boost (week 24). Samples (more ...)
In order to corroborate the trends in the relative CD4+/CD8+ contributions to the T-cell responses observed from the limited cohort sizes described above, we collected data from several other immunization studies and summarize these results in Fig. along with those described above. Homologous immunization with Ad5-gag at 109 and 1011 viral particle doses (administered at weeks 0, 4, and 24) elicited antigen-specific T-lymphocyte populations with CD8+ T-cell compositions ranging from 73% to 99% (mean ± standard error, 90% ± 2%); the CD8+ composition was not influenced by the dose used. In contrast, priming immunizations with DNA-CRL1005 produced a highly variable (2% to 84%) but generally balanced CD8+/CD4+ distribution (mean ± standard error, 51% ± 7.25%). The administration of an Ad5-gag boost to the same animals resulted in Gag-specific T-cell populations that were 35% to 97% CD8+ (mean ± standard error, 85% ± 4%).
FIG. 8. Percentages of Gag-specific T cells that are CD3+ CD8+ in rhesus macaques immunized with various regimens. The adenovirus type 5 prime-adenovirus type 5 boost (Ad5-Ad5) cohort consisted of animals given two priming doses of the vector (more ...)