Potential To Streamline Heterologous DNA Prime and NYVAC/Protein Boost HIV Vaccine Regimens in Rhesus Macaques by Employing Improved Antigens

Abstract

In a follow-up to the modest efficacy observed in the RV144 trial, researchers in the HIV vaccine field seek to substantiate and extend the results by evaluating other poxvirus vectors and combinations with DNA and protein vaccines. Earlier clinical trials (EuroVacc trials 01 to 03) evaluated the immunogenicity of HIV-1 clade C GagPolNef and gp120 antigens delivered via the poxviral vector NYVAC. These showed that a vaccination regimen including DNA-C priming prior to a NYVAC-C boost considerably enhanced vaccine-elicited immune responses compared to those with NYVAC-C alone. Moreover, responses were improved by using three as opposed to two DNA-C primes. In the present study, we assessed in nonhuman primates whether such vaccination regimens can be streamlined further by using fewer and accelerated immunizations and employing a novel generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher expression levels and more balanced immune responses. Three different DNA-C prime/NYVAC-C+ protein boost vaccination regimens were tested in rhesus macaques. All regimens elicited vigorous and well-balanced CD8(+)and CD4(+)T cell responses that were broad and polyfunctional. Very high IgG binding titers, substantial antibody-dependent cellular cytotoxicity (ADCC), and modest antibody-dependent cell-mediated virus inhibition (ADCVI), but very low neutralization activity, were measured after the final immunizations. Overall, immune responses elicited in all three groups were very similar and of greater magnitude, breadth, and quality than those of earlier EuroVacc vaccines. In conclusion, these findings indicate that vaccination schemes can be simplified by using improved antigens and regimens. This may offer a more practical and affordable means to elicit potentially protective immune responses upon vaccination, especially in resource-constrained settings.

Importance: Within the EuroVacc clinical trials, we previously assessed the immunogenicity of HIV clade C antigens delivered in a DNA prime/NYVAC boost regimen. The trials showed that the DNA prime crucially improved the responses, and three DNA primes with a NYVAC boost appeared to be optimal. Nevertheless, T cell responses were primarily directed toward Env, and humoral responses were modest. The aim of this study was to assess improved antigens for the capacity to elicit more potent and balanced responses in rhesus macaques, even with various simpler immunization regimens. Our results showed that the novel antigens in fact elicited larger numbers of T cells with a polyfunctional profile and a good Env-GagPolNef balance, as well as high-titer and Fc-functional antibody responses. Finally, comparison of the different schedules indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination with protein may be very efficient, thus showing that the novel antigens allow for easier immunization protocols.

FIG 1

Immunization schedules for comparison of the three different priming regimens. Three groups of 8 macaques each were immunized two or three times with DNA (light blue) and twice with NYVAC/protein (dark blue). At the indicated time points, blood was collected for ELISpot analysis, intracellular cytokine staining (ICS), or antibody analysis (red symbols).

FIG 2

All vaccination regimens induced similarly large numbers of IFN-γ-secreting T cells. Freshly isolated PBMCs collected at the indicated time points (weeks after the start of the study) were stimulated with nine different peptide pools and subjected to IFN-γ ELISpot analysis. The total numbers of spot-forming units (SFUs) per million cells for group A (3xD_4w; red), group B (3xD_2w; blue), and group C (2xD_4w; green) are shown, along with medians and interquartile ranges. (A) Responses after DNA prime immunizations. (B) Responses after NYVAC/protein boosting (note the different scales). Data sets obtained 2 weeks after the last DNA immunization and the last NYVAC/protein boost are highlighted by a shaded background.

FIG 3

All vaccine groups demonstrated similar T cell responses, characterized by a balanced ratio of CD4 to CD8 cells and large proportions of polyfunctional cells. PBMCs from vaccinated macaques were isolated at week 26, stimulated with the 9 peptide pools, stained for CD3, CD4, and CD8 and for the intracellular cytokines IFN-γ, IL-2, and TNF by use of fluorescently labeled antibodies, and analyzed by flow cytometry. (A) Percentages of CD4⁺ and CD8⁺ HIV-specific T cells (sums for all peptide pools and cytokines; medians with quartiles are shown). (B) CD4⁺ (upper graph) and CD8⁺ (lower graph) HIV-specific T cells separated for determination of cytokine release profiles (medians with quartiles are shown). Pie charts below show the relative fractions of trifunctional (green), bifunctional (blue), and monofunctional (red) T cells for the three groups. (C) Pie charts showing relative fractions of CD4⁺ (left) and CD8⁺ (right) T cells stimulated by the indicated peptide pools (sums for all cytokine profiles).

FIG 4

All vaccination regimens induced strong IgG antibody responses against several Env antigens, as well as V1V2-specific responses and responses against p24 and p66(RT). IgG binding magnitudes are indicated as AUC values, and medians and interquartile ranges are shown for the specified time points postvaccination. Shaded areas highlight time points 2 weeks after completion of the DNA and NYVAC/protein immunizations. (A) Time course for antibody responses against consensus clade C gp140. (B) Responses against gp70V1V2. (C) Responses against the other tested proteins, as indicated.

FIG 5

Comparison of antibody responses against the different antigens at weeks 26 (A) and 40 (B). IgG binding magnitude data are from Fig. 4.

FIG 6

The vaccination regimens elicited only negligible serum IgA responses. IgA binding magnitudes are indicated by MFI, and medians and interquartile ranges are shown for each time point postvaccination. (A) Responses against consensus clade C gp140 and 1086 gp120. (B) Responses against the indicated proteins.

FIG 7

Sera from immunized monkeys show poor neutralizing activity against a panel of Env isolates in a TZM-bl cell assay. Pseudoviruses carrying the indicated Env isolates were incubated with sera from the immunized monkeys before addition of TZM-bl cells. The serum dilution leading to a 50% decrease of luciferase activity (IC₅₀) is shown for values above the assay's threshold of 20. (A) Time course of IC₅₀ values over the study duration for the highly neutralization-sensitive tier 1A clade C isolate MW965.26 (top left) and for the other tested Env proteins, as indicated. (B) Week 26 IC₅₀ values for all tested envelopes. MuLV was used as a negative control.

FIG 8

Sera from immunized monkeys show poor neutralizing activity in an A3R5 cell assay. The graphs show time courses of serum neutralization activity in an A3R5 cell assay against infectious molecular clones carrying envelope proteins from the indicated isolates.

FIG 9

Plasmas from immunized monkeys show moderate ADCC and ADCVI activities. (A) ADCC activity. CEM.NKR_CCR5 target cells coated with gp120 of isolate 1086 (left) or TV1 (right) were mixed with PBMCs at an E:T ratio of 30:1. A granzyme B substrate and dilution series of the plasma samples were added, and the cells were incubated and assessed by flow cytometry for viable cells showing granzyme B activity. The antibody titer was calculated by interpolating the reciprocal plasma dilution causing a granzyme B activity matching the cutoff value of the assay. Horizontal arrows mark titers exceeding the upper limit of the assay (102,400). Diamonds indicate titers significantly above the week 0 values, and asterisks indicate significant between-group differences (P < 0.05, with appropriate Bonferroni correction). Time points 2 weeks after the last applications of DNA and NYVAC/protein are highlighted by shading. (B) ADCVI activity. CEM.NKR_CCR5 target cells were infected with HIV-1 DU156 or DU422 for 3 days. Plasma samples obtained at week 26 and PBMCs at an E:T ratio of 10:1 were added. After 8 days, virus amounts were measured by p24 ELISA, and the % inhibition was calculated as the proportion of p24 obtained in the presence of plasma samples versus week 0 control plasma.