This phase 1 study tested the safety and immunogenicity of a DNA plasmid and recombinant MVA-vectored vaccines, both encoding defined HIV-1 CTL epitopes within Env, Gag, Nef, Pol, Rev, and Vpr genes that are restricted by the HLA-A2, -A3, and -B7 HLA superfamily allelic products, defined HTL epitopes of HIV-1 within Env, Gag, Pol, and Vpr genes, and the PADRE HTL epitope. This represents the first clinical trial of these second-generation multiepitope vaccines. The prime-boost vaccination design was chosen in part because of the low level of immunogenicity in a previous clinical trial of a DNA plasmid-vectored vaccine, EP HIV-1090, that encoded only the CTL epitopes and PADRE, and was administered alone (28
). Another clinical trial that was ongoing at the time our study was planned, included administration of the same HIV-1 HTL epitopes expressed as a recombinant polypeptide protein given alone or in combination with the previously tested EP HIV-1090 DNA plasmid-vectored vaccine (38
). The recombinant polypeptide protein induced polyfunctional CD4+
helper T-cell responses in two-thirds of subjects after two immunizations, but the EP HIV-1090 vaccine was minimally immunogenic, failing to reproducibly induce either CD4+
T-cell responses (38
EP-1233 and MVA-mBN32 in our study were both safe and well tolerated with more local injection site reactogenicity, and more adverse events were observed after administration of the MVA-mBN32 than the EP-1233 DNA plasmid-vectored vaccines. There were no proven episodes of myopericarditis and no cardiotoxicity in association with the MVA-BN vaccine. Although myopericarditis cannot be eliminated as a cause, the QTc interval prolongation after MVA vaccination in one subject may have been related to trazodone, bupropion, or methamphetamine use reported by the subject (17
). Amphetamines are associated with acute and chronic cardiotoxicities, but unlike trazodone and bupropion, do not appear on lists of drugs that cause QTc interval prolongation. Myocarditis is a cause of QTc interval prolongation (50
); however, this subject did not have other reasons to invoke this as the cause. Systemic reactions were infrequent and mild in the vaccine group, and not different comparing the DNA plasmid-vectored vaccine to the MVA-BN32 vaccine, but were more common than among the placebo control recipients. The subcutaneous nodules at the MVA-mBN32 injection site were a different phenomenon than injection site induration, resolved more slowly, were clinically not very significant and have not been reported in studies employing other MVA vaccines.
A low rate of IL-2- and IFN-γ-secreting CD4+ and CD8+ T-cell responses was detected by ICS in the present study; all four positive specimens from the three vaccine responders had T-cell responses to the CTL peptide pool manifested by IL-2, IFN-γ, and TNF-α production and perforin by the CD8+ T cells. One participant's cells from one time point produced IL-2, IFN-γ, and TNF-α in response to the HTL peptide pool and PADRE. Cells from one subject whose CD8+ T cells produced IFN-γ, TNF-α, and perforin in response to the CTL peptide pool in the ICS assay also produced IFN-γ to one HIV-1 Gag peptide that is a CTL epitope in the ELISPOT assay.
Low levels of CTL and HTL responses induced by this prime-boost vaccination regimen were not due to lack of an immune response to MVA. Antibodies to MVA-BN were induced by the MVA-mBN32 vaccine. Also, the historical and clinical assessments to exclude persons who previously had been vaccinated with vaccinia virus were in general successful since only two vaccine recipients and two placebo recipients had detectable antibody at low titer to MVA at day 0. The subjects with detectable anti-MVA antibody before vaccination did not have T-cell cytokine responses in the ICS assay. Antibodies to MVA were boosted to the highest level after the second MVA-mBN32 vaccination compared to after the first and waned to levels similar to those after the first vaccination by 3 months after the second vaccination.
Clinical studies in the literature have reported use of the plasmid DNA/recombinant MVA prime-boost vaccination strategy consisting of other constructs expressing HIV-1 proteins with demonstration of a good safety profile, lack of cases of myopericarditis and various rates of CD4+
T-cell immune responses to the HIV-1 components (13
). The dose, route of administration, quality of immune priming achieved with the DNA plasmid vector, sensitivity of the assays used to measure vaccine-induced T-cell responses, and time after booster immunization when peak response is measured all may affect observed response rates to the prime-boost vaccination regimens of between about 10 and 92% (27
). Previous immune status to vaccinia virus did not appear to affect responses to HIV-1 proteins expressed by at least one candidate MVA vaccine (29
The low level of immunogenicity associated with these vaccines is similar to that of others based largely on the use of defined CTL epitopes (13
). We included several design features which differentiate the product tested here and which we believed would increase vaccine potency. These features were the selection of the highest affinity HLA-supertype epitopes, the use of spacers to optimize epitope processing and presentation, the inclusion of the universal PADRE HTL epitope as an adjuvant and formulation with PVP to augment cellular uptake of the plasmid DNA. Despite these features the EP-1233 DNA failed to efficiently prime immune responses.
We now believe the observed poor levels of immunogenicity in our study and others using epitope-based vaccines may well reflect a suboptimal match between the vaccine design and the DNA and MVA vectors with respect to how the epitopes are delivered to the immune system. Most recent experimental evidence indicates a dominant role for the cross-presentation pathway following intramuscular vaccine immunogen delivery using DNA plasmid vectors, although some data support a minor role for direct antigen presentation in vivo
by transfected myocytes (14
). Vaccinia viruses and MVA can infect professional antigen-presenting cells, such as dendritic cells, and thus may be able to mediate vaccine immunogen presentation through the direct priming pathway. However, this infection can impact dendritic cell maturation and function, and evidence points to the dominant use of the cross-presentation pathway for the induction of cellular immune responses to the transgene and viral proteins for vaccinia virus vaccines and MVA-based vectors (20
). A recent report of high levels of HIV-1-specific CD4+
(77%) and CD8+
(42%) T-cell responses to other DNA plasmid and recombinant MVA-vectored vaccines that encode noninfectious virus-like particles of HIV-1 and were administered in a heterologous prime-boost regimen as in our study supports the need for emphasis on optimizing the HIV-1 insert to achieve a desirable cellular immune response (26
Efficient induction of cellular immune responses mediated using cross-presentation appears to require stable protein antigen as the substrate and accumulation of amounts sufficient to induce responses, whereas direct presentation is efficient when unstable or rapidly degraded proteins are presented (46
). The EP1233 and the MVA-mBN32 vaccines in our study both encode unnatural genes composed of CTL and HTL epitopes separated by spacer sequences designed to optimize intracellular processing of individual epitopes within transfected or infected cells (5
). Although it is feasible that this design could support efficient induction of CTL via the transfer of proteasome substrates (46
), the results from our clinical trial indicate otherwise. Overall, our current understanding of the DNA and MVA vaccine vectors and this phase 1 clinical trial result suggest that these vector platforms are not well-suited for use with T-cell epitope-based vaccines (9
). Vaccine delivery methods that efficiently deliver epitope-based vaccines to professional antigen presenting cells will need to be investigated to further develop the concept of epitope-based vaccines.
In summary, our study demonstrated higher reactogenicity and adverse event rates with MVA-mBN32 compared to the DNA plasmid-vectored vaccine, but the MVA was still well-tolerated. These vaccines given in high dose and prime-boost sequence did not elicit adequate HIV-1-specific T-cell immune responses and further optimization of the HIV-1 T-cell epitope-based constructs expressed by DNA plasmids and MVA-vectored vaccines is needed.