Streamlining Heterologous DNA-Prime and NYVAC / Protein-Boost HIV Vaccine Regimens in 1 Rhesus Macaques by Employing Improved Antigens 2 3

36 In follow up to the modest efficacy observed in the RV144 trial, the HIV vaccine field seeks to 37 substantiate and extend the results by evaluating other poxvirus vectors and combinations with 38 DNA and protein vaccines. Earlier clinical trials (EuroVacc 01 – 03) evaluated the 39 immunogenicity of HIV-1 C-clade GagPolNef and gp120 antigens delivered via the poxviral 40 vector NYVAC. These showed that a vaccination regimen including DNA-C-priming prior to a 41 NYVAC-C boost considerably enhanced vaccine-elicited immune responses as compared to 42 NYVAC-C alone. Moreover, responses were improved using three as opposed to two DNA-C 43 primes. Here, we assessed in non-human primates whether such vaccination regimens can be 44 further streamlined using fewer and accelerated immunizations when employing a novel 45 generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher 46 expression levels and more balanced immune responses. Three different DNA-C prime / 47 NYVAC-C + protein boost vaccination regimens were tested in rhesus macaques. All regimens 48 elicited vigorous and well balanced CD8+ and CD4+ T cell responses that were broad and 49 polyfunctional. Very high IgG binding titers, substantial ADCC and modest ADCVI activities, 50 but very low neutralization activity were measured after final immunizations. Overall, immune 51 responses elicited in all three groups were very similar and of greater magnitude, breadth and 52 quality as compared to earlier generation EuroVacc vaccines. In conclusion, these findings 53 indicate that vaccination schemes can be simplified using improved antigens and regimens. This 54 may offer a more practical and affordable means to elicit potentially protective immune responses 55 upon vaccination especially in resource-constrained settings. 56 57 IMPORTANCE 58 Within the EuroVacc clinical trials we previously assessed the immunogenicity of HIV C-clade 59 antigens delivered in a DNA-prime/NYVAC-boost regimen. The trials showed that the DNA 60 prime crucially improved the responses and three DNA primes with NYVAC-boost appeared 61 optimal. Nevertheless, T cell responses were primarily Env-directed and humoral responses 62 modest. The aim of this study was to assess improved antigens for their capacity to elicit more 63 potent and balanced responses in rhesus macaques even when using various simpler 64 immunization regimens. Our results showed that the novel antigens in fact elicited higher 65 numbers of T cells with a polyfunctional profile and good Env:GagPolNef-balance, as well as 66 high titer and Fc-functional antibody responses. Finally, comparison of the different schedules 67 indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination 68 with protein may be very efficient, thus showing that the novel antigens allow for easier 69 immunization protocols. 70 71 72 INTRODUCTION 73 In order to develop an efficacious prophylactic vaccine against infection with human 74 immunodeficiency virus (HIV-1), various approaches are being pursued to optimize the immune 75 functions that might contribute to protection from infection or disease. Several factors are likely 76 important to consider for the potential success of a vaccine. Besides the choice of the antigen as 77 core component of any vaccine, the mode of delivery, the immunization regimen, route, and dose 78 as well as the exploitation of immune-modulating factors either added in trans as adjuvants or 79 representing intrinsic properties of e.g. vector systems may impact efficacy. Current approaches 80 are mainly focused on the induction of antibody responses as they are considered to prevent 81 infection, while cytotoxic CD8+ T lymphocyte (CTL) responses are generally thought to modify 82 disease progression by reducing viral load (1). However, recent studies of rhesus macaques 83 immunized with a novel cytomegalovirus vector indicate the potentially protective role of CD8 84 T cells, especially those of effector memory phenotype (2–4). Moreover, given that helper CD4+ 85 T cell responses are important for high-quality B cell responses, a vaccine candidate should likely 86 elicit responses of all kinds – innate, B cell, helper T cell and CTL – in a balanced manner. 87 The 31 % protection observed in the RV144 Thai trial (5), which used a combination of the 88 poxvirus ALVAC expressing Gag, Pro, and gp120-TM as prime and AIDSVAX B/E gp120 as 89 boost came as a surprise, as the AIDSVAX itself lacked efficacy (6, 7). This finding highlights 90 the potential value of replication-deficient live recombinant viral vectors and heterologous prime91 boost regimens to elicit protective immune responses. In particular, priming with DNA-vectored 92 vaccines prior to the application of the viral vector, mostly employing adenoviruses or 93 poxviruses, has repeatedly been shown to considerably increase cellular and humoral immune 94 responses as compared to the viral vector alone (8–10). In the context of the EuroVacc clinical 95 trials EV01 and EV02 (11, 12), we tested HIV C-clade antigens (GagPolNef + gp120) delivered 96 via the poxvirus NYVAC with or without a DNA-prime encoding the same set of antigens. These 97 vectors previously showed promising immunogenicity profiles in preclinical assays and 98 protective efficacy in primates against SHIV89.6 challenge (13, 14). The clinical trials 99 demonstrated that the vaccine candidates were safe and well tolerated and that DNA-priming 100 dramatically improved the T cell responses elicited by NYVAC. Both the proportion of 101 responders and the number of HIV-specific T cells as measured by ELISpot-analysis for IFN-γ 102 secreting cells increased twoto four-fold (12, 15). Other studies have shown even better 103 augmentation (16). The T cell responses were mainly of the CD4 phenotype and directed against 104 Env, although a balanced response against all antigens and a balanced ratio of CD4 and CD8 105 responses might be desirable, especially against Gag, as CD8 T cell responses against this antigen 106 are associated with long-term disease control in some patients (17, 18). Regarding the 107 immunization schedule, a regimen consisting of three DNA-primes (at month 0, 1 and 2 plus a 108 single NYVAC-boost at month 6 (using the same generation of DNA and NYVAC as in EV01 109 and EV02) was superior to two DNA-primes at month 0 and 1 followed by two NYVAC-boosts 110 at month 5 and 6 (EV03-study, (19)). Although macaques seem to be more responsive in 111 immunogenicity analyses as compared with humans, the rhesus macaque model allows at least a 112 ranking of the immunogenicity of different vectors (20). Therefore, despite additional limitations 113 such as differences in the configuration of MHC-I loci (21), monkey trials are instrumental to 114 evaluate the numerous vaccine regimens and help in the design of human clinical trials. 115 Importantly, the cellular immune responses observed in humans in the EV02 study were 116 remarkably similar to those obtained in macaques immunized with the same DNA and NYVAC 117 vaccine candidates and according to the same regimen (22), which supports the value of the 118 rhesus macaque model. 119 The HIV vaccine field plans to substantiate and extend the results observed in RV144 by 120 evaluating other poxvirus vectors, such as NYVAC in combination with DNA and protein 121 vaccines. A key question that has to be addressed is how to best schedule the immunizations. 122 This is an especially important issue, as complex immunization regimens comprising many 123 immunizations (for instance 7 in the AIDSVAX trials, and 6 in RV144) are unlikely to be 124 brought to market. Yet, simple modifications in number, timing, or sequence of heterologous 125 vectors can have a major impact on immunogenicity (23, 24). Such modifications offer a lot of 126 potential as they can be tested quite easily without again performing safety analyses if the 127 individual components have already been assessed. Therefore, the present study is designed to 128 determine whether an accelerated DNA schedule or fewer DNA injections elicit equally effective 129 priming responses if optimized vaccines are employed. Toward this goal, both the DNA and 130 NYVAC vaccines were redesigned and thus represent a new generation of vaccine candidates 131 with optimized antigens and vectors. 132 Specifically, the major optimization comprises modified antigens, mainly in regard to the weak 133 Gag-specific responses observed in NHP and clinical studies (EV01 – EV03) (11, 12, 15, 19). 134 For this, the original GagPolNef antigen, consisting of a 160 kDa fusion protein with 135 modifications introduced for increased safety (i.e. abrogated myristoylation, lack of IN, 136 inactivation of PR, splitting of RT and scrambling of Nef (25)) was refined further to allow for 137 efficient production and release of virus-like particles and to better balance the relative 138 expression of Gag and PolNef antigens. For plasmid-delivery of these next generation antigens, 139 Gag and PolNef were split on two DNA vectors in order to reduce the individual plasmid size, 140 thus likely facilitating uptake in vivo. Moreover, a gp140 form was used instead of gp120 to more 141 closely resemble the native trimeric envelope structure (26), and was included as a third DNA 142 vector. Immunogenicity analyses in mice clearly showed superiority of this three-plasmid 143 configuration compared to the parental EuroVacc vaccines both regarding the magnitude of 144 antigen-specific T cells, as well as the balance towards the different antigens (27). Improved 145 responses after splitting of a different Gag-Pol-Nef antigen into separate parts have also been 146 observed in humans in clinical phase I trials (16, 28). For the immunological assessment of our 147 next generation antigens in rhesus macaques, the plasmid backbone was also changed to VRC148 8400 (29) from the pORT constructs (30) to fu