Implantation of reporter-labeled tumor cells in an immunocompetent host involves a risk of their immune elimination. We have studied this effect in a mouse model of breast cancer after the orthotopic implantation of mammary gland adenocarcinoma 4T1 cells genetically labelled with luciferase (Luc). Mice were implanted with 4T1 cells and two derivative Luc-expressing clones 4T1luc2 and 4T1luc2D6 exhibiting equal in vitro growth rates. In vivo, the daughter 4T1luc2 clone exhibited nearly the same, and 4T1luc2D6, a lower growth rate than the parental cells. The metastatic potential of 4T1 variants was assessed by magnetic resonance, bioluminescent imaging, micro-computed tomography, and densitometry which detected 100-μm metastases in multiple organs and bones at the early stage of their development. After 3–4 weeks, 4T1 generated 11.4 ± 2.1, 4T1luc2D6, 4.5 ± 0.6; and 4T1luc2, <1 metastases per mouse, locations restricted to lungs and regional lymph nodes. Mice bearing Luc-expressing tumors developed IFN-γ response to the dominant CTL epitope of Luc. Induced by intradermal DNA-immunization, such response protected mice from the establishment of 4T1luc2-tumors. Our data show that natural or induced cellular response against the reporter restricts growth and metastatic activity of the reporter-labelled tumor cells. Such cells represent a powerful instrument for improving immunization technique for cancer vaccine applications.

quinone oxidoreductase (Nqo1) and heme oxygenase 1 (HO-1), indicating the induction of oxidative stress response. The capacity to induce oxidative stress and stress response appeared to be an intrinsic property of a vast variety of RTs: enzymatically active and inactivated, bearing mutations of drug resistance, following different routes of processing and presentation, expressed from viral or synthetic expression-optimized genes. The total ROS production induced by RT genes of the viral origin was found to be lower than that induced by the synthetic/expression-optimized or chimeric RT genes. However, the viral RT genes induced higher levels of ROS production and higher levels of HO-1 mRNA than the synthetic genes per unit of protein in the expressing cell. The capacity of RT genes to induce the oxidative stress and stress response was then correlated with their immunogenic performance. For this, RT genes were administered into BALB/c mice by intradermal injections followed by electroporation. Splenocytes of immunized mice were stimulated with the RT-derived and control antigens and antigen-specific proliferation was assessed by IFN-γ/IL-2 Fluorospot. RT variants generating high total ROS levels induced significantly stronger IFN-γ responses than the variants inducing lower total ROS, while high levels of ROS normalized per unit of protein in expressing cell were associated with a weak IFN-γ response. Poor gene immunogenicity was also associated with a high (per unit of protein) transcription of antioxidant response element (ARE) dependent phase II detoxifying enzyme genes, specifically HO-1. Thus, we have revealed a direct link between the propensity of the microbial proteins to induce oxidative stress and their immunogenicity.

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.

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DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid (“surrogate challenge”). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199–220 and aa 528–543. Drug-resistance mutations disrupted the epitope at aa 205–220, while the CTL epitope at aa 202–210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.