We hypothesized that vigorous replication of a pathogen may be critical for eliciting the 2 most potent and balanced immune response against it. Hence, attenuation/inactivation 3 (as in conventional vaccines) should be avoided. Instead, necessary safety should be 4 provided by placing replication of the pathogen under stringent control and of activating 5 time-limited replication of the pathogen strictly in an administration region in which 6 pathology cannot develop. Immunization will then occur in the context of highly efficient 7 pathogen replication and uncompromised safety. We found that localized activation in 8 mice of efficient but limited replication of a replication-competent controlled herpesvirus 9 vector resulted in a greatly enhanced immune response to the virus or an expressed 10 heterologous antigen. This finding supports the above hypothesis as well as suggests 11 that the vectors may be promising novel agents worth exploring for the 12 prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in 13 particular in immunocompromised patients.
that attenuated replicating viruses induce more complete and more potent immune 1 responses to autologous or heterologous antigens than corresponding non-replicating 2 viruses. 3 We hypothesized that a virus vector that could replicate in a controlled fashion with 4 nearly the same efficiency as the respective wildtype virus (referred to herein as 5 "replication-competent controlled virus vector") would induce an even more potent and 6 complete immune response to itself or an expressed heterologous antigen than a 7 corresponding attenuated vector (26). Our hypothesis is in part based on the rational 8 expectation that an efficiently replicating virus will produce a stronger inflammatory 9 response than an attenuated virus, which inflammatory response will result in a potent 10 activation of the innate immune system and, consequently, in strong and lasting B and T 11 cell responses (27) . To realize such an immunization strategy, a regulation system must 12 be employed that reliably and stringently controls viral replication as well as is capable 13 of being turned on and off at will. However, virus disseminates after administration. 14 Simply restricting the number of replication cycles will not be enough: depending on the 15 number of cycles allowed, there will be a more or less pronounced manifestation of the 16 toxicity typical for the viral vector used. For example, allowing an HSV-1 vector to 17 replicate for a certain period of time may cause unacceptable neurotoxicity. Hence, the 18 regulation system must be capable of exerting regional control over viral replication so 19 that the immunizing virus only replicates in a locale in which it is certain not to cause a 20 disease phenotype. HSV-GS7 replication was also tightly controlled in vivo, two of three groups of mice 8 were administered HSV-GS7 virus (50,000 pfu per mouse) to the footpad, and the mice 9 of one of the latter groups were given ulipristal intraperitoneally (i,p.) as well as, 3 h 10 later, were subjected to a heat treatment to the footpads at 45 0 C for 10 min. One day 11 later, all mice were euthanized, and DNA and RNA were extracted from their feet and 12 analyzed by qPCR and RT-qPCR, respectively. Substantially larger amounts of HSV-1 13 DNA were detected in the feet of heat/ulipristal-treated mice than in not-treated mice 14 (Fig. 1D ). Expression of several viral genes was observed for activated virus but not for 15 unactivated virus, strongly suggesting that viral replication and gene expression only 16 occurred subsequent to heat/ulipristal activation.
It is noted that stocks of the recombinant viruses were prepared in cells that were 18 subjected to daily heat treatment in the presence of antiprogestin until maximal 19 cytopathic effect was reached. Titrations of the viruses were carried out on cells that 20 provided missing proteins in trans.
Protective immunity induced by activated HSV-GS3. Induction of protective 1 immunity was evaluated in a mouse footpad lethal challenge model (35). In a first 2 experiment, virus vectors were administered under anesthesia to the plantar surfaces of 3 both rear feet of adult Swiss Webster outbred female mice (50,000 pfu per animal; 20 4 animals per group). Concurrently, and again 24 h later, the animals of one group 5 received an intraperitoneal injection of 0.5 mg/kg of mifepristone. Three hours after 6 inoculation, the mice of the latter group were subjected to heat treatment (43.5 0 C for 30 7 min) by immersion of their hindfeet in a temperature-controlled water bath. Twenty-two 8 days later, all animals were challenged by a 20-fold lethal dose of HSV-1 wildtype strain 9 17syn+ administered by the same route as the original virus inoculum. Survival of the 10 animals was followed until no more lethal endpoints were reached, i.e., until all surviving 11 animals had fully recovered (Fig. 2) . ICP4(-) replication-incompetent HSV-1 12 recombinant KD6 (36) induced a modest level of immunity. As had been expected, 13 because it did not replicate and also did not express the major transcriptional regulator 14 ICP4, unactivated HSV-GS3 provided a comparable degree of protection. Activated To determine whether immunization with activated HSV-GS3 reduced replication of the 22 challenge virus more effectively than unactivated HSV-GS3 or replication-defective KD6 23 virus, additional groups of animals (5 animals per group) were immunized and 1 challenged by wildtype virus as described above. Four days after challenge, the animals 2 were euthanized, feet were dissected and homogenized, and virus present in the 3 homogenates was titrated. Results revealed that activated HSV-GS3 virus reduced 4 challenge virus replication by nearly two orders of magnitude (Table 1) . Unactivated Second immunization further enhances protective immunity. We next investigated 11 whether a second activation treatment applied two days after the first treatment (i.e., at Table 2 . Results of a meta-analysis of the HSV-GS7 data 8 are presented graphically in Fig. 6 . The difference in immunization efficacy between 9 activated and unactivated HSV-GS7 was found to be highly significant. Also significant 10 was the increase in protection afforded by second immunization with an activated HSV-11 GS7 vector. The effect of second activation two days after virus administration and first 12 activation was not statistically relevant. Second activation tended to modestly enhance 13 protective immunity in a majority of experiments in which this was addressed. However, 14 in some experiments, e.g., in the experiment reported in Fig. 5A , an effect was not 15 apparent. It is noted that the conditions for second activation had not been optimized. subjected to activation treatment. Both HA RNA and protein levels appeared to be 2 somewhat higher in twice-activated animals than in once-activated animals.
To assess immune responses (three weeks after immunization), additional groups of 4 mice were inoculated with saline (mock immunization), or with 50,000 pfu of HSV-GS3 5 or HSV-GS11 (three groups). As in the above-described part of the experiment, all 6 animals in the HSV-GS3 group as well as in two of the HSV-GS11 groups were 7 subjected to an activation treatment. The animals of one of the latter groups received a 8 second activation treatment two days later. Serum samples were tested for their ability 9 to neutralize EIV Prague/56. As expected, neutralizing antibodies were not detected in 10 unimmunized (not shown), mock-immunized or vector-immunized animals (Fig. 8D ).
Unactivated HSV-GS11 was capable of inducing a neutralizing antibody response.
Activation of HSV-GS11 shortly after inoculation resulted in a several-fold magnified 13 response. It is noted that twice-activated HSV-GS11 elicited an only marginally better 14 response than once-activated virus. HA-specific T cells present in PBMCs were 15 quantified by the same type of responder cell frequency assay that had been used to 16 assess numbers of HSV-1-specific T cells. HA-specific T cells were not detected in 17 unimmunized (not shown) or mock-immunized animals (Fig. 8E ). Induction of a T cell Generalizing these findings, it appears that replication-competent controlled vectors are 12 promising new agents that may better protect against diseases caused by the viruses 13 from which they were derived than replication-incompetent/inactivated vaccines and, 14 possibly, also live attenuated vaccines. What is perhaps even more important, our 15 observations suggest that replication-competent controlled vectors can be excellent 16 immunization platforms that may be exploited for the elaboration of new "vaccines". distinguish the possibilities that the second activation was only marginally effective 7 because the immune system was already maximally engaged by the initial immunization 8 or because optimal conditions for the second activation treatment had not been 9 identified. Additional research is required to resolve this question.
Immunization by replication-competent controlled vectors represents a novel paradigm 11 that may be elaborated in various ways. Therefore, the work presented herein should be In this report, we have focused on immunization effects of once-activated replication-10 competent controlled vectors that undergo one round of replication. We note that viral 11 vectors that were intended to be limited to one cycle of replication were advanced 12 before as potential vaccines (45). These so-called disabled infectious single-cycle 17GS43 virion DNA. Subsequent to the addition of mifepristone to the medium, the co-transfected cells were exposed to 43.5 0 C for 30 min and then incubated at 37 0 C. 1 Subsequently, on days 2 and 3, the cells were again incubated at 43.5 0 C for 30 min and 2 then returned to 37 0 C. Picking and amplification of plaques, screening and plaque 3 purification was performed essentially as described for HSV-GS3 (3,34). The resulting Neutralizing antibody assay. After collection, the blood was allowed to clot for 30 min. Table 3 . the proteins were used to coat a 96 well ELISA plate which was allowed to air-dry. Table   21 2 and Fig. 6 for comparable experiments. for each HSV-GS3 or HSV-GS7 group; n = 10 for mock; *** p ≤ 0.05). See Table 2 Fig. 6 for comparable experiments. ("activated + boost"; n = 10), and activated on day 1, readministered 50,000 pfu/mouse 10 of HSV-GS7 21 days after the first inoculation and reactivated 3 h later ("activated + 11 boost + 2nd activation"; n = 10). Twenty-one days after the last treatment, all mice were 12 challenged with a 20-fold lethal dose of wild-type HSV-1 strain 17syn+ applied to both 13 rear feet. ** p ≤ 0.05; *** p < 0.01. (B) A similar experiment except that both initial and 14 second immunizations were with 5,000 pfu/mouse of HSV-GS7 (n = 10; for second 15 immunization n = 20; *** p ≤ 0.05). The data are presented as percent survival for each 16 treatment group. See Table 2 and Fig. 6 for comparable experiments. Vector replication was activated in some treatment groups by administration of heat and 20 ulipristal as described in Fig. 3 . One treatment group received a second activation 21 treatment that was administered two days after the first activation treatment. Mice feet 22 were harvested 24 hours after the last treatment, and RNA was isolated and cDNA prepared. Samples were analyzed by qPCR with EIV Prague/56 HA-specific Taqman 1 primers/probe (Table 3) 