Previous prime-boost clinical studies of candidate T cell vaccines for HIV have suggested that adenovirus vectors were more immunogenic than poxvirus vectors in terms of cellular immune responsiveness. In the EV02 trial, we demonstrate that DNA plus NYVAC is a highly immunogenic prime-boost regimen, with durable responses up to 1 yr after vaccination. The primary analysis of the vaccine-induced T cell responses was performed using a validated IFN-γ ELISPOT assay. Polychromatic flow cytometry was used to characterize the phenotypic and the functional profiles of the vaccine-induced T cell populations. Furthermore, both the IFN-γ ELISPOT assay and polychromatic flow cytometry were instrumental in analyzing the breadth of the vaccine-induced T cell responses and for performing epitope mapping. All the analyses have been performed on frozen blood mononuclear cell samples, and the evaluation of the immunogenicity has been assessed in the IFN-γ ELISPOT assay after a conventional overnight stimulation of the blood mononuclear cells, with the panel of peptide pools encompassing Env, Gag, Pol, and Nef of HIV-1 clade C CN54.
The majority (18 out of 20, 90%) of the volunteers immunized with the DNA C plus NYVAC C regimen had a positive response using the IFN-γ ELISPOT assay at weeks 26 or 28, the primary endpoints of the study. The percentage of responders in the NYVAC C–alone group was 33%. These results were superior to previous studies that have evaluated the immunogenicity of DNA plus poxvirus (MVA) vaccine regimens in which the percentage of responders was 17% (low dose DNA) and ~40% (high dose DNA) when conventional overnight stimulation was performed to analyze IFN-γ–secreting cells (as used in EV02) (16
). Responses were observed in the eight volunteers in the high dose DNA plus MVA study only using a cultured ELISPOT assay (5-d stimulation) able to detect weak vaccine-induced T cell responses (16
). The immunogenicity observed in EV02 was also higher than that observed in studies using Ad5-based vaccine strategies and in the same range of studies using DNA plus Ad5 regimens (unpublished data).
Vaccine-induced IFN-γ–secreting cells were detected only in 7 out of 20 volunteers after DNA C vaccination and before NYVAC C boosting. However, despite the fact that vaccine-induced T cell responses were not measurable in the majority of volunteers after the two DNA vaccinations, the DNA priming was clearly responsible for the substantial increase in the immunogenicity and magnitude (see below) of the vaccine-induced T cell responses in the DNA C plus NYVAC C group compared with the NYVAC C–alone group. These results indicate that the assessment of immunogenicity after immunization with DNA alone is not a reliable measure of the priming ability of DNA candidate vaccines.
Collectively, these results indicate that the DNA C plus NYVAC C regimen is superior to the NYVAC C–alone regimen and has a degree of immunogenicity comparable to that of other promising candidate vaccines that have entered large phase II/IIB clinical studies (3
The magnitude of the vaccine-induced T cell responses as measured by the frequency of IFN-γ–secreting cells using the ELISPOT assay was substantially higher (three- to fourfold) in the DNA C plus NYVAC C group compared with the NYVAC C–alone group. The magnitude of the vaccine-induced T cell responses observed after DNA C plus NYVAC C immunization was superior to that observed in previous DNA plus MVA studies (17
) and comparable to that of Ad5 candidate vaccines and to the DNA plus Ad5 vaccine combination (unpublished data).
The vaccine-induced T cell responses were predominantly (in 100% of responders) mediated by CD4 T cells. However, CD8 T cell responses were also found in 47% of responders. Furthermore, both vaccine-induced CD4 and CD8 T cell responses were predominantly directed against Env (the median magnitude of Env T cell responses in the DNA C plus NYVAC C group vs. the NYVAC C–alone group was 299 and 131 SFU/106 cells, respectively). T cell responses against Gag, Pol, and Nef were detected in 48% of volunteers and had lower magnitude (the median magnitude of Gag-, Pol-, and Nef-specific T cell responses was ~100 SFU/106 cells).
The finding of predominant CD4 T cell responses after immunization with DNA C plus NYVAC C is consistent with previous studies (3
) investigating the T cell responses induced by poxvirus candidate vaccines. The predominant CD4 T cell response after immunization with vaccines including poxviruses seems to differentiate these latter from Ad5-based candidate vaccines and the DNA plus Ad5 vaccine regimen that appear to induce more balanced CD4 and CD8 T cell responses (unpublished data). Three factors may have contributed to the dominant CD4 T cell and Env-specific responses: (a) the DNA priming that seems to favor the development of the CD4 T cell response; (b) the monovalent nature of the NYVAC C construct in which Env, Gag, Pol, and Nef are expressed within the same vector; and (c) the construction of the NYVAC vector with regard to Env, which is expressed in a secreted form. The use of multivalent instead of monovalent DNA and Ad5-based vaccines may reduce the Env immunodominance in favor of more balanced Env, Gag, and Pol responses. The secreted form of Env is likely to be responsible for favoring the exogenous pathway of antigen presentation and thus stimulation of CD4 T cells.
In natural infection studies, it has been reported that Gag-specific CD8 T cell responses are associated with better control of HIV disease in individuals with chronic HIV-1 infection, whereas Env-specific CD8 T cell responses are associated with poor control (25
). However, it is not clear from this study whether (a) Gag-specific CD8 T cell responses are the cause or rather the effect of lower levels of virus replication, and (b) results generated in the nonhuman primate model have shown that the presence of Env- and Gag-specific vaccine-induced T cell responses in animals immunized with DNA plus Ad5 expressing Env and Gag conferred better protection from disease after infection compared with animals immunized with DNA plus Ad5 Gag-expressing vaccines (26
The DNA C plus NYVAC C vaccine regimen induced CD4 T cell responses in 100% of the immunized individuals and CD8 T cell responses in 50% of vaccines. In this regard, it is important to mention that the presence of the vaccine-induced CD4 T cell responses strongly correlated with the containment of viremia in macaques exposed to the highly pathogenic SIVmac251
after immunization with the DNA-SIV-gag-env
). Therefore, vaccine-induced CD4 T cell responses may be effective in the attenuation of HIV disease.
Env- and Gag-specific CD4 and CD8 T cell responses induced by DNA C plus NYVAC C vaccination were polyfunctional. Recently (8
), the term polyfunctional has been used to define T cell responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, and MIP-1β, as well as cytotoxic activity, comprise T cell populations also able to secrete IL-2 and retain antigen-specific proliferation capacity, whereas the term “only-effector” defines T cell responses/populations with typical effector functions but lacking IL-2 and proliferation capacity. Of interest, several studies (11
) have demonstrated that polyfunctional and not only-effector T cell responses were associated with protective antiviral immunity (32
). The DNA C plus NYVAC C immunization therefore induced the best functional profile of virus-specific CD4 and CD8 T cells capable of controlling virus replication in several chronic virus infections, such as cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and HIV-1 in patients with nonprogressive disease (8
). These data are consistent with our previous study demonstrating that vaccine-induced CD8 T cells after vaccination with DNA C plus NYVAC C were highly polyfunctional and that almost 75% of these cells had four or five functions based on IFN-γ, TNF-α, MIP-1β, IL-2, and CD107a (34
The polyfunctional vaccine-induced CD4 T cell populations were CD45RA−
, a phenotype of cells with effector functions but also with the ability to secrete IL-2 and endowed with proliferation capacity (20
). The phenotype of the vaccine-induced CD4 T cells remained unchanged over time. The vaccine-induced CD8 T cells were either CD45RA−
. This phenotypic profile defines memory CD8 T cells at intermediate and advanced stages of differentiation and is consistent with our recent study using CD27 and CD45RO (34
). The presence and rapid appearance after immunization of CD45RA+
CD8 T cells is of interest. This phenotype has been proposed to define effector CD8 T cells at advanced stages of differentiation (21
). Furthermore, virus-specific CD45RA+
CD8 T cells have been found in controlled chronic virus infections such as CMV and EBV (21
), and a correlation between the percentage of this cell population and virus control has also been shown in HIV-1 infection (8
). The majority of the vaccine-induced CD45RA−
CD8 T cell population disappeared over time, whereas ~90% of vaccine-induced CD8 T cells were CD45RA+
at week 48, 6 mo after the last immunization. Therefore, the DNA C plus NYVAC C vaccination likely induces the generation of a long-lived population of memory CD8 T cells.
Of interest, the DNA C plus NYVAC C vaccination induced a broad T cell response, with a mean 4.2 epitopes recognized per volunteer. About 50% of epitopes identified in the EV02 study have not previously been described according to the Los Alamos database (24
), and approximately two out of three of the total epitopes identified were located in constant regions.
A critical component of the effectiveness of vaccines is their ability to induce long-lasting immunity. The results obtained in EV02 study after vaccination with DNA C plus NYVAC C are extremely promising. More than 70% of volunteers have still measurable vaccine-induced T cell responses 1 yr after the last vaccination and, more importantly, the magnitude of the T cell responses is not substantially changed compared with 1 mo after the completion of the vaccination regimen.
Poxvirus vectors have traditionally represented an interesting platform in the HIV vaccine arena because of (a) their large and successful use in the veterinary field, (b) extensive safety data in humans, (c) their use in vaccine platforms for other infectious diseases and cancer, and (d) their facility to be manipulated and to be inserted with large gene fragments (4
). However, disappointing results on their immunogenicity in humans had seriously cast doubt on the validity of the poxvirus platform and the rationale for their further development in the HIV vaccine arena. The present data generated within the EV02 study have clearly demonstrated that a poxvirus candidate vaccine, NYVAC, in combination with DNA is highly immunogenic, induces vigorous and broad T cell responses, comprising of both CD4 and CD8 T cell responses, which are polyfunctional, and more importantly, this vaccine regimen induces long-lasting T cell immunity.
These promising results support the further development of the poxvirus platform and the move of the DNA C plus NYVAC C vaccine regimen into larger clinical trials.