MVA-CMDR was safe and well tolerated at all dosages and routes tested. There were no serious adverse events related to the vaccine and there was no evidence of cardiac toxicity. MVA-CMDR elicits a modest, yet readily detectable cellular immune response against the HIV insert gene products in most vaccine recipients. Using traditional assay platforms, such as lymphocyte proliferation or chromium release assays, up to 100% of the volunteers had detectable lymphoproliferative responses (depending upon dose and route) and two-thirds (63%) had CD8-dependent CTL activity depending upon the dose and route. These data are consistent with the priming of both CD4+ and CD8+ T cells. Using more sophisticated flow cytometry-based assay techniques the balance and frequency of the cellular immune response is further elucidated. CD4+ T cells, in particular those targeting the Env protein, were most frequently detected with both whole blood ICS and multi-functional ICS. The high-dose IM vaccination regimen resulted in the induction of CD4+ T cells targeting Env in almost all volunteers using both ICS assays. However, CD8+ T cells specific for Env were detected in only 30% of the same volunteers using the two assay platforms. The whole blood ICS assay appeared to be more sensitive for detecting Gag-specific CD8+ T cells – 40% versus no positive responses with the multifunctional assay in the high-dose IM group. This observation may represent an important qualitative difference in sensitivity between the two assay platforms. The detection of Env-specific CD4+ T cells that synthesized IL-2, IFNγ and TNFα by flow cytometry is consistent with the lymphocyte proliferation data. The sporadic detection of insert-specific CD8+ T cells by 51Cr-release and flow cytometry is consistent with the induction of a low frequency of CD8+ T cells with high proliferative capacity. The durability of the cellular immune response is reflected in the 100% lymphoproliferative response rates in both IM route groups to the TH023 recombinant protein at 6 months after completion of the vaccination schedule and in the detection of Elispot positive responses at the same time-point. Overall, Env was consistently the predominant target of the cellular immune response and CD4+ T cells were the most frequently detected responder cell type. A dose and route effect of the vaccination procedure was observed with the high-dose (108 pfu) IM route of vaccine delivery being the most immunogenic.
The observed trend towards elicitation of numerically greater CD4+
T cell responses targeting HIV-1 Envelope by the vaccine is an important observation. In humans, the use of recombinant multigenic MVA as a vector for both priming and boosting immune responses appears to be similar to multigenic DNA vaccines, which also induce primarily CD4+
T cell responses against Env-derived gene products 
. Both the present study and the EV02 study of a NYVAC-C vaccine product by the EuroVacc Consortium suggest that vaccination with vaccinia-derived poxvirus vectors may be equivalent in their ability to induce predominantly Env-specific CD4+
T cells with multigenic products 
. While canarypox-based vaccines have been shown to induce CD4+
T cells responses against Envelope antigens in a multigenic canarypox-prime and protein-boost setting, it is as yet unclear whether the phenomenon of Env-specific CD4+
T cell dominance can be generalized to all multigenic poxvirus vectors 
. In contrast, adenovirus-based vectors used either as a prime and boost, or as a boost for a DNA vaccine prime, appear to generate higher levels of cellular immune responses in general and generate a better balance of CD4+
T cell responses to multiple gene products in a multigenic setting 
. While studies performed in the setting of HIV-1 infection have shown that robust, polyfunctional CD8+
T cell responses targeting multiple epitopes in the Gag (and in some cases Nef) protein are associated with better clinical outcome and protection from disease progression it is unclear whether such cells would also provide protection from infection 
. In fact, in the setting of a vaccine that induced Gag- and Nef-specific CD4+
T cell responses no protection from HIV-infection was conferred 
. Hence, the functional relevance of Env (and Gag) specific CD4+
T cells in setting of vaccination for prevention of infection needs to be further explored. In particular, subtle differences in the qualities of T cells generated in response to different vector-based delivery combinations of multigenic gene products should be the focus of future studies. The ability to generate T cells that either help appropriate antibody class switching and affinity maturation, or act as direct effector cells, or both, will be a critical quality of any effective HIV vaccine.
Binding antibody responses were detected against both p24 and gp120. A clear dose- and route-dependence of the binding antibody response was evident with the IM routes being most immunogenic. Antibody titers peaked two-weeks after vaccination and waned, but were still detectable at 6 months post-vaccination. The diminution of binding antibody GMTs in the interim between 2 weeks and 6 months post-vaccination completion may be important with respect to poxvirus vectors in general. In the ALVAC-HIV/AIDSVAX B/E Phase III trial (RV144) the modest protective efficacy appeared be transient during the 6 months immediately post-vaccination and then waned. If induction of transient antibody responses is typical of non-replicating poxvirus vectors in general then strategies that increase the durability of such responses will need to be established. MVA-CMDR would therefore be an ideal candidate for testing different prime/boost strategies that will increase the durability of antibody responses. These antibodies likely had functional capacity as measured by ADCC activity in four volunteers in the high-dose IM group following three immunizations with MVA-CMDR. Previously, 4 immunizations with canarypox (vCP1521) expressing CRF01_AE gp120 had failed to induce ADCC activity 
, implying that MVA-CMDR is a more potent inducer of ADCC than canarypox vectors expressing CRF01_AE antigens. It is unknown whether such antibody responses would, on their own, be sufficient to mitigate transmission or to reduce viral set point and/or modulate disease progression.
Compared with other stand alone MVA-based HIV vaccine products MVA-CMDR would appear to be at least as immunogenic as the ADMVA 
and the TBC-M4 
vaccine products (at a 2.5 fold lower dose than either) and substantially more immunogenic than the HIVA vaccine product 
. MVA-CMDR also appears to be at least as immunogenic as the related attenuated poxvirus vector NYVAC product vP2010 
. For HIV and other infectious diseases, it has become increasing commonplace to use rMVA and NYVAC products to augment DNA primed cellular and humoral responses 
. MVA-CMDR has been shown previously to be an effective boost for cellular immune responses generated by a heterologous DNA vaccine prime 
. When used as stand-alone products recombinant poxvirus vectors induce quantitatively lower insert-directed cellular immune responses compared with recombinant Adenovirus-based vectors 
. Their continued use as HIV vaccine modalities is likely to be contingent upon: (i) successful pairing with other vectors in prime-boost regimens to increase the magnitude of the immune response; (ii) verification that the immune responses generated are qualitatively different to those generated by alternative vectors and (iii) successful demonstration of vaccine-induced protection in primate lentivirus challenge models.
MVA-CMDR was designed to be administered in combination with other vaccine products - either as the prime for a prime/boost poxvirus followed by recombinant protein vaccine schedule, as performed in the recently completed RV144 trial, or in combination with other vaccines such as DNA-based or Adenovirus-based HIV vaccines. Given that DNA-prime and MVA-boost vaccinations are superior to MVA alone vaccinations in animal models and in human trials 
, and the fact that MVA-CMDR has already been used successfully to boost a heterologous DNA-based vaccine product in humans 
it is likely that this product will prove useful as a boost for cellular responses induced by other heterologous priming modalities. In addition, the versatility of MVA-CMDR may be further explored by exploiting its capacity to prime for antibody responses in prime/boost strategies that further test the concepts arising from the RV144 trial