Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC

Abstract

As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here, we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a nonhuman primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140, and Gag/Gag-Pol-Nef-derived virus-like particles (VLPs) from clade C and were used as the prime, with recombinant virus plus envelope protein used as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial.IMPORTANCE Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.

Keywords: Gag-Pol-Nef; HIV; NYVAC; NYVAC-KC; T cell response; antibody responses; gp140; nonhuman primates; vaccines.

FIG 1

Immunization schedule for the virus prime and virus/protein boost regimen. Two groups of 8 macaques were each immunized twice with virus alone (either NYVAC-C-KC or NYVAC-C), followed by three immunizations with virus plus protein. All immunizations were by the intramuscular (i.m.) route. Blood was collected for ELISpot analysis or antibody analysis at the indicated time points.

FIG 2

The replication-competent NYVAC-C-KC induces a greater T cell response does replication-deficient NYVAC-C. PBMCs were freshly isolated at the indicated time points and restimulated with a pool of peptides in an ELISpot assay. The number of spot-forming cells (producing IFN-γ) per 10⁶ cells is shown. (A) Each value is the sum of the responses from the nine peptides per each of the 8 animals. Statistical significance was determined using the Mann Whitney method, with a confidence interval of 95%. w, week. (B) The number of animals in each group (n = 8) that were responders is shown for each time point tested. Criteria for responders were defined as ≤50 SFC/10⁶ cells and a 4-fold increase above the baseline level for any pool of peptides. Immunizations were at weeks 0, 4, 12, 24, and 49 (indicated by arrows). Statistical significance was determined using a Wilcoxon rank sum test (A) and by a Fisher exact test (B).

FIG 3

The replication-competent NYVAC-C-KC (group 1) induces more polyfunctional CD4⁺ and CD8⁺ T cell responses than replication-deficient NYVAC-C (group 2). PBMCs were obtained at the indicated time points, stimulated with the nine peptide pools, and stained for CD3, CD4, and CD8, as well as intracellular IFN-γ, IL-2, and TNF-α. Responding CD4⁺ (A and B) and CD8⁺ (C and D) T cells were quantified by flow cytometry. (A) The fraction of triple-positive (red) responses for NYVAC-C-KC vaccinees (group 1) was greater at every time point. (B) Responses at weeks 8 and 28 were significantly higher for NYVAC-C-KC vaccinees than for NYVAC-C vaccinees (group 2). (C) The fraction of triple-positive (red) responses for NYVAC-C-KC vaccinees was greater at every time point. (D) Though not statistically significant, the responses at nearly all time points were higher for those animals vaccinated with NYVAC-C-KC than with NYVAC-C. Statistics calculated by Wilcoxon signed rank test using SPICE software (NIAID).

FIG 4

NYVAC-C-KC (group 1) gave improved IgG antibody responses to HIV-1 antigens and both groups had low responses to IgA. (A) A customized binding antibody multiplex assay was used to quantify IgG antibodies that bound to antigen-coated beads. The binding response for each antigen per each animal is shown as the area under the curve (AUC) of the titration curve, and medians and interquartile ranges are shown as horizontal lines. (B) Comparative responses of the week 14 and week 51 sera toward all antigens assessed. Statistical significance was determined using a Wilcoxon rank sum test (for panels A and B). (C) IgA responses of the week 51 sera toward all antigens assessed.

FIG 5

Antibody response to the V1-V2 region was strong for both NYVAC-C-KC and NYVAC-C groups. Response was measured by an ELISA, and the results for dilutions of 1:2430 (A) and 1:7290 (B) are shown. At both dilutions, the antibody levels following the week 24 boost were significantly higher for NYVAC-C-KC (group 1) than for NYVAC-C (group 2). P values were calculated by the Wilcoxon rank-sum test. OD, optical density.

FIG 6

Neutralizing antibody panel potency subject-specific and average magnitude breadth curves for TZM-bl assay. A magnitude-breadth (MB) curve of 50% inhibitory dose (ID₅₀) neutralizing antibody titers against pseudoenvelopes shown over time (x axis, neutralization titers; y axis, fraction of viruses neutralized). Isolates were the following: MN.3, MW965.26, and TV1.21 for week 14; TV1.21, BaL.26, Bx08.16, MN.3, MW965.26, SF162.LS, SHIV SF162P4, SHIV-SF162.P3, SS1196.1, SHIV 1157ipEL-p, SHIV 1157ipd3N4, and SHIV BAL-P4 for week 26; MN.3 and MW965.26 for week 48; TV1.21, BaL.26, Bx08.16, MN.3, MW965.26, SF162.LS, SHIV SF162P4, SHIV-SF162.P3, and SS1196.1 for week 51. Values of <20 (limit of detection of the assay) were assigned a value of 10. Dashed lines represent subject-specific responses. Solid lines represent group averages. P values were derived from a Wilcoxon rank sum test.

FIG 7

Macaque sera neutralization in TZM-bl assay. (A) Serum samples were diluted and tested for neutralization against the indicated pseudoviruses. The value shown is the reciprocal of dilution at which 50% inhibition (ID₅₀) was observed for each animal; median and interquartile range are shown at the indicated time points of the pseudoviruses carrying MW965.26 or MN.3. Statistical significance was determined using a Mann-Whitney method. (B) ID₅₀ values of the serum samples from week 26 and week 51 for pseudoviruses carrying Envs from the indicated isolates.

FIG 8

Serum samples from macaques collected at week 51 demonstrated a strong HIV-1 response in both ADCC and ADCVI assays. (A) ADCC assay. CEM.NKRCCR5 target cells coated with TV1 gp120 were coincubated with PBMC preparations (including NK cells) at an effector/target ratio of 30:1 in the presence of plasma dilutions from the immunized macaques. The amount of granzyme B released by NK cells was measured using a fluorescent substrate. The plasma dilution was calculated to match the granzyme B activity cutoff, and the reciprocals are given as titers for values above the assay’s cutoff value of 100, with median and interquartile range also shown. (B) ADCVI assay. CEM.NKRCCR5 cells were infected with infectious HIV-1 strain DU156 or DU422 and then coincubated with PBMC effector cells in the presence of plasma at an effector/target ratio of 10:1. The amount of virus released was measured by a p24 ELISA, and the inhibition of virus production compared to that of samples lacking plasma is given as a percentage.