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. IMPORTANCEWithin 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.

HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates

“…Thus, the replication-competent NYVAC-C-KC vector consists of two NYVAC-KC viruses that express different clade C HIV-1 antigens under the control of the same synthetic early/late poxvirus promoter (55), one (NYVAC-KC-gp140) expressing Env gp140 from strain 96ZM651 and the other (NYVAC-KC-Gag-Pol-Nef) expressing Gag from strain 96ZM651 and Pol and Nef from strain CN54. A description of the HIV-1 gp140 and Gag-Pol-Nef sequences included in the NYVAC-KC recombinant vectors was previously reported (10,24). Next, the VACV B19R gene (B18R in the WR strain) was deleted from the replication-competent recombinant vectors NYVAC-KC-gp140 and NYVAC-KC-Gag-Pol-Nef to generate the replication-competent recombinant NYVAC-KC-gp140-ΔB19R and NYVAC-KC-Gag-Pol-Nef-ΔB19R vectors, respectively, according to the same methodology as the one described previously (19).…”

“…The overall strength of the HIV-1-specific T cell immune responses induced was analyzed by an IFN-␥ ELISpot analysis using freshly isolated PBMCs obtained from each immunized rhesus macaque, as previously described (10). Briefly, PBMCs were stimulated in triplicate with HIV-1 Env, Gag, Pol, and Nef peptide pools at 1 g/ml or with phytohemagglutinin (PHA) (2.5 g/ml) as a positive control, while the addition of medium only served as a negative control, according to protocols described previously (10). SFUs were counted as a measure of the magnitude of HIV-1-specific T cell responses.…”

“…At the same time, these constructs maintained limited virus spread in tissues and an attenuated phenotype (26). Additionally, further improved NYVAC recombinant vectors expressing HIV-1 immunogens, such as HIV-1 clade C(ZM96) trimeric soluble gp140 or Gag(ZM96)-Pol-Nef(CN54) as Gag-derived virus-like particles (VLPs), have been shown to have an enhanced HIV-1-specific immunogenicity profile in mice (24) and nonhuman primates (NHPs) (10,13).…”

“…Thus, in order to optimize the immunization protocol with NYVAC vectors expressing HIV-1 antigens, various approaches in NHPs have been evaluated, either comparing NYVAC to ALVAC (13) or combining NYVAC with DNA vectors (10), peptides (22), and dendritic cell targets (28), which have all demonstrated promising results.…”

Application of area scaling analysis to identify natural killer cell and monocyte involvement in the GranToxiLux antibody dependent cell‐mediated cytotoxicity assay

“…We have previously described a high‐throughput quantitative flow cytometry‐based assay that allows for detection of the proteolytic activity of Granzyme B (GzB) at the single cell level to identify target cells that have received a cytotoxic hit as a result of antigen‐specific antibody‐Fc receptor interactions . We and others have broadly applied this assay, the ADCC‐GranToxiLux assay (ADCC‐GTL), to the measure of HIV‐ or SIV‐specific antibody responses generated by natural infection or by active and passive immunization strategies .…”