PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice.

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.

SAMHD1 Promotes the Antiretroviral Adaptive Immune Response in Mice Exposed to Lipopolysaccharide.

SAMHD1 is a potent HIV-1 restriction factor that blocks reverse transcription in monocytes, dendritic cells and resting CD4+ T cells by decreasing intracellular dNTP pools. However, SAMHD1 may diminish innate immune sensing and Ag presentation, resulting in a weaker adaptive immune response. To date, the role of SAMHD1 on antiretroviral immunity remains unclear, as mouse SAMHD1 had no impact on murine retrovirus replication in prior in vivo studies. Here, we show that SAMHD1 significantly inhibits acute Friend retrovirus infection in mice. Pretreatment with LPS, a significant driver of inflammation during HIV-1 infection, further unmasked a role for SAMHD1 in influencing immune responses. LPS treatment in vivo doubled the intracellular dNTP levels in immune compartments of SAMHD1 knockout but not wild-type mice. SAMHD1 knockout mice exhibited higher plasma infectious viremia and proviral DNA loads than wild-type mice at 7 d postinfection (dpi), and proviral loads inversely correlated with a stronger CD8+ T cell response. SAMHD1 deficiency was also associated with weaker NK, CD4+ T and CD8+ T cell responses by 14 dpi and weaker neutralizing Ab responses by 28 dpi. Intriguingly, SAMHD1 influenced these cell-mediated immune (14 dpi) and neutralizing Ab (28 dpi) responses in male but not female mice. Our findings formally demonstrate SAMHD1 as an antiretroviral factor in vivo that could promote adaptive immune responses in a sex-dependent manner. The requirement for LPS to unravel the SAMHD1 immunological phenotype suggests that comorbidities associated with a "leaky" gut barrier may influence the antiviral function of SAMHD1 in vivo.

C(3)1-TAg in C57BL/6 J background as a model to study mammary tumor development.

Diagnosis and prognosis of breast cancer is based on disease staging identified through histopathological and molecular biology techniques. Animal models are used to gain mechanistic insights into the development of breast cancer. C(3)1-TAg is a genetically engineered mouse model that develops mammary cancer. However, carcinogenesis caused by this transgene was characterized in the Friend Virus B (FVB) background. As most genetic studies are done in mice with C57BL/6 J background, we aimed to define the histological alterations in C3(1)-TAg C57BL/6 J animals. Our results showed that C3(1)-TAg animals with C57BL/6 J background develop solid-basaloid adenoid cystic carcinomas with increased fibrosis, decreased area of adipocytes, and a high proliferative index, which are triple-negative for progesterone, estrogen, and human epidermal growth factor receptor 2 (HER2) receptors. Our results also revealed that tumor development is slower in the C57BL/6 J background when compared with the FVB strain, providing a better model to study the different stages in breast cancer progression.

A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells.

PD-1-targeted therapies have shown modest antiviral effects in preclinical models of chronic viral infection. Thus, novel therapy protocols are necessary to enhance T cell immunity and viral control to overcome T cell dysfunction and immunosuppression. Here, we demonstrate that nanoparticle-based therapeutic vaccination improved PD-1-targeted therapy during chronic infection with Friend retrovirus (FV). Prevention of inhibitory signals by blocking PD-L1 in combination with therapeutic vaccination with nanoparticles containing the microbial compound CpG and a CD8+ T cell Gag epitope peptide synergistically enhanced functional virus-specific CD8+ T cell responses and improved viral clearance. We characterized the CD8+ T cell populations that were affected by this combination therapy, demonstrating that new effector cells were generated and that exhausted CD8+ T cells were reactivated at the same time. While CD8+ T cells with high PD-1 (PD-1hi) expression turned into a large population of granzyme B-expressing CD8+ T cells after combination therapy, CXCR5-expressing follicular cytotoxic CD8+ T cells also expanded to a high degree. Thus, our study describes a very efficient approach to enhance virus control and may help us to understand the mechanisms of combination immunotherapy reactivating CD8+ T cell immunity. A better understanding of CD8+ T cell immunity during combination therapy will be important for developing efficient checkpoint therapies against chronic viral infections and cancer.IMPORTANCE Despite significant efforts, vaccines are not yet available for every infectious pathogen, and the search for a protective approach to prevent the establishment of chronic infections, i.e., with HIV, continues. Immune checkpoint therapies targeting inhibitory receptors, such as PD-1, have shown impressive results against solid tumors. However, immune checkpoint therapies have not yet been licensed to treat chronic viral infections, since a blockade of inhibitory receptors alone provides only limited benefit, as demonstrated in preclinical models of chronic viral infection. Thus, there is a high interest in the development of potent combination immunotherapies. Here, we tested whether the combination of a PD-L1 blockade and therapeutic vaccination with functionalized nanoparticles is a potent therapy during chronic Friend retrovirus infection. We demonstrate that the combination therapy induced a synergistic reinvigoration of the exhausted virus-specific CD8+ T cell immunity. Taken together, our results provide further information on how to improve PD-1-targeted therapies during chronic viral infection and cancer.

B-Cell Control of Regulatory T Cells in Friend Virus Infection.

B lymphocytes have well-established effector roles during viral infections, including production of antibodies and functioning as antigen-presenting cells for CD4+ and CD8+ T cells. B cells have also been shown to regulate immune responses and induce regulatory T cells (Tregs). In the Friend virus (FV) model, Tregs are known to inhibit effector CD8+ T-cell responses and contribute to virus persistence. Recent work has uncovered a role for B cells in the induction and activation of Tregs during FV infection. In addition to inducing Tregs, B cell antibody production and antigen-presenting cell activity is a target of Treg suppression. This review focuses on the dynamic interactions between B cells and Tregs during FV infection.

Immunization with a murine cytomegalovirus based vector encoding retrovirus envelope confers strong protection from Friend retrovirus challenge infection.

Immunization vectors based on cytomegalovirus (CMV) have attracted a lot of interest in recent years because of their high efficacy in the simian immunodeficiency virus (SIV) macaque model, which has been attributed to their ability to induce strong, unusually broad, and unconventionally restricted CD8+ T cell responses. To evaluate the ability of CMV-based vectors to mediate protection by other immune mechanisms, we evaluated a mouse CMV (MCMV)-based vector encoding Friend virus (FV) envelope (Env), which lacks any known CD8+ T cell epitopes, for its protective efficacy in the FV mouse model. When we immunized highly FV-susceptible mice with the Env-encoding MCMV vector (MCMV.env), we could detect high frequencies of Env-specific CD4+ T cells after a single immunization. While the control of an early FV challenge infection was highly variable, an FV infection applied later after immunization was tightly controlled by almost all immunized mice. Protection of mice correlated with their ability to mount a robust anamnestic neutralizing antibody response upon FV infection, but Env-specific CD4+ T cells also produced appreciable levels of interferon γ. Depletion and transfer experiments underlined the important role of antibodies for control of FV infection but also showed that while no Env-specific CD8+ T cells were induced by the MCMV.env vaccine, the presence of CD8+ T cells at the time of FV challenge was required. The immunity induced by MCMV.env immunization was long-lasting, but was restricted to MCMV naïve animals. Taken together, our results demonstrate a novel mode of action of a CMV-based vaccine for anti-retrovirus immunization that confers strong protection from retrovirus challenge, which is conferred by CD4+ T cells and antibodies.

Friend retrovirus studies reveal complex interactions between intrinsic, innate and adaptive immunity.

Approximately 4.4% of the human genome is comprised of endogenous retroviral sequences, a record of an evolutionary battle between man and retroviruses. Much of what we know about viral immunity comes from studies using mouse models. Experiments using the Friend virus (FV) model have been particularly informative in defining highly complex anti-retroviral mechanisms of the intrinsic, innate and adaptive arms of immunity. FV studies have unraveled fundamental principles about how the immune system controls both acute and chronic viral infections. They led to a more complete understanding of retroviral immunity that begins with cellular sensing, production of type I interferons, and the induction of intrinsic restriction factors. Novel mechanisms have been revealed, which demonstrate that these earliest responses affect not only virus replication, but also subsequent innate and adaptive immunity. This review on FV immunity not only surveys the complex host responses to a retroviral infection from acute infection to chronicity, but also highlights the many feedback mechanisms that regulate and counter-regulate the various arms of the immune system. In addition, the discovery of molecular mechanisms of immunity in this model have led to therapeutic interventions with implications for HIV cure and vaccine development.

Fcγ Receptor Type I (CD64)-Mediated Impairment of the Capacity of Dendritic Cells to Activate Specific CD8 T Cells by IgG-opsonized Friend Virus.

Dendritic cells (DCs) express Fcγ receptors (FcγRs) for the binding immune complexes (ICs) consisting of IgG and antigens (Ags). IC⁻FcγR interactions have been demonstrated to enhance activation and antigen-presenting functions of DCs. Utilizing Friend virus (FV), an oncogenic mouse retrovirus, we investigated the effect of IgG-opsonization of retroviral particles on the infection of DCs and the subsequent presentation of viral antigens by DCs to virus-specific CD8 T cells. We found that opsonization by virus-specific non-neutralizing IgG abrogated DC infection and as a consequence significantly reduced the capacity of DCs to activate virus-specific CD8 T cells. Effects of IgG-opsonization were mediated by the high-affinity FcγR type I, CD64, expressed on DCs. Our results suggest that different opsonization patterns on the retroviral surface modulate infection and antigen-presenting functions of DCs, whereby, in contrast to complement, IgG reduces the capacity of DCs to activate cytotoxic T cell (CTL) responses.

Effects of Friend Virus Infection and Regulatory T Cells on the Antigen Presentation Function of B Cells.

Friend virus (FV) is a naturally occurring mouse retrovirus that infects dividing cells of the hematopoietic lineage, including antigen-presenting cells (APCs). The infection of APCs by viruses often induces their dysfunction, and it has been shown that FV infection reduces the ability of dendritic cells (DCs) to prime critical CD8+ T cell responses. Nonetheless, mice mount vigorous CD8+ T cell responses, so we investigated whether B cells might serve as alternative APCs during FV infection. Direct ex vivo analysis of B cells from FV-infected mice revealed that infected but not uninfected B cells upregulated expression of the costimulatory molecules CD80, CD86, and CD40, as well as major histocompatibility complex class II (MHC-II) molecules. Furthermore, in vitro studies showed that, compared to uninfected B cells from the same mice, the FV-infected B cells had significantly enhanced APC function, as measured by their capacity to prime CD8+ T cell activation and proliferation. Thus, in contrast to DCs, infection of B cells with FV enhanced their APC capacity and ability to stimulate the CD8+ T cell responses essential for virus control. FV infections also induce the activation and expansion of regulatory T cells (Tregs), so it was of interest to determine the impact of Tregs on B cell activation. The upregulation of costimulatory molecule expression and APC function of B cells was even more strongly enhanced by in vivo depletion of regulatory T cells than infection. Thus, Tregs exert potent homeostatic suppression of B cell activation that is partially overcome by FV infection.IMPORTANCE The primary role of B cells in immunity is considered the production of pathogen-specific antibodies, but another, less-well-studied, function of B cells is to present foreign antigens to T cells to stimulate their activation and proliferation. Dendritic cells (DCs) are considered the most important antigen-presenting cells (APCs) for CD8+ T cells, but DCs lose APC function when infected with Friend virus (FV), a model retrovirus of mice. Interestingly, B cells were better able to stimulate CD8+ T cell responses when they were infected with FV. We also found that the activation status of B cells under homeostatic conditions was potently modulated by regulatory T cells. This study illustrates an important link between B cell and T cell responses and illustrates an additional mechanism by which regulatory T cells suppress critical T cell responses during viral infections.

Friend retrovirus infection induces the development of memory-like natural killer cells.

Traditionally, NK cells belong to the innate immune system and eliminate virus-infected cells through their germline-encoded receptors. However, NK cells were recently reported to possess memory-like functions that were predominantly provided by hepatic NK cells. Memory properties were mainly documented in contact hypersensitivity models or during cytomegalovirus infections. However, the precise role and the physiologic importance of memory-like NK cells during retroviral infections are still under investigation. Here, we show that Friend retrovirus (FV) infection of mice induced a population of phenotypically memory-like NK cells at 28 days post infection. Upon secondary antigen encounter, these NK cells showed an increased production of the pro-inflammatory cytokines IFNγ and TNFα as well as the death ligand FasL in comparison to naïve NK cells. Furthermore, we found an augmented elimination of antigen-matched but not antigen-mismatched target cells by these memory-like NK cells. In adoptive cell transfer experiments, equal antiviral activities of splenic and hepatic memory-like NK cells during the late phase of acute FV infection were found. Our results strongly imply the existence and antiviral activity of spleen and liver memory-like NK cells in FV infection, which efficiently respond upon secondary exposure to retroviral antigens.

The Cytotoxic Activity of Natural Killer Cells Is Suppressed by IL-10+ Regulatory T Cells During Acute Retroviral Infection.

Natural killer (NK) cells play a key role in host defense against cancer and viral infections. It was shown that NK cells are important for the control of acute retroviral infections, but their antiviral activity depends on multiple parameters such as viral inoculation dose, interactions with myeloid cell types and the cytokine milieu. In addition, during an ongoing retroviral infection regulatory T cells (Tregs) can suppress NK cell functions. However, the precise role of Tregs on the initial NK cell response and their immediate antiviral activity after an acute retroviral infection is still unknown. Here we show that thymus-derived Tregs suppress the proliferation, effector functions and cytotoxicity of NK cells very early during acute Friend Retrovirus (FV) infection. Tregs exhibited an activated phenotype and increased the production of the immunosuppressive cytokines IL-10 and TGF-ß after FV infection of mice. Neutralization of the immunosuppressive cytokine IL-10 resulted in a significant augmentation of NK cell functions. Although the activation of dendritic cells (DCs) and macrophages as well as the IL-15 cytokine levels were increased after Treg depletion, Tregs mainly affect the NK cell activity in an IL-10-regulated pathway. In this study we demonstrate an IL-10-dependent suppression of NK cells by activated Tregs during the first days of a retroviral infection.

Regulatory T cells suppress virus-specific antibody responses to Friend retrovirus infection.

Recent vaccine studies with experimental antigens have shown that regulatory T cells (Tregs) constrain the magnitude of B cell responses. This homeostatic Treg-mediated suppression is thought to reduce the potential of germinal center (GC) responses to generate autoreactive antibodies. However, essentially opposite results were observed in live influenza infections where Tregs promoted B cell and antibody responses. Thus, it remains unclear whether Tregs dampen or enhance B cell responses, especially during live viral infections. Here, we use mice infected with Friend retrovirus (FV), which induces a robust expansion of Tregs. Depletion of Tregs led to elevated activation, proliferation, and class switching of B cells. In addition, Treg depletion enhanced the production of virus-specific and virus-neutralizing antibodies and reduced FV viremia. Thus, in contrast to influenza infection, Tregs either directly or indirectly suppress B cells during mouse retroviral infection indicating that the ultimate effect of Tregs on B cell responses is specific to the particular infectious agent.

Regulatory T cells constrain the TCR repertoire of antigen-stimulated conventional CD4 T cells.

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non-self-antigen, a TCRß transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T-cell repertoire was used. The response of EF4.1 mice to an I-Ab-associated epitope of the F-MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg-depleted EF4.1 mice were immunized, and the extent of the Vα2-bearing, antigen-specific TCR repertoire was characterized by high-throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre-immune repertoire. Injection of anti-CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non-self-antigens.

Dose of Retroviral Infection Determines Induction of Antiviral NK Cell Responses.

Natural killer (NK) cells are part of the innate immune system and recognize virus-infected cells as well as tumor cells. Conflicting data about the beneficial or even detrimental role of NK cells in different infectious diseases have been described previously. While the type of pathogen strongly influences NK cell functionality, less is known about how the infection dose influences the quality of a NK cell response against retroviruses. In this study, we used the well-established Friend retrovirus (FV) mouse model to investigate the impact of virus dose on the induction of antiviral NK cell functions. High-dose virus inoculation increased initial virus replication compared to that with medium- or low-dose viral challenge and significantly improved NK cell activation. Antiviral NK cell activity, including in vivo cytotoxicity toward infected target cells, was also enhanced by high-dose virus infection. NK cell activation following high-dose viral challenge was likely mediated by activated dendritic cells (DCs) and macrophages and the NK cell-stimulating cytokines interleukin 15 (IL-15) and IL-18. Neutralization of these cytokines decreased NK cell functions and increased viral loads, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we demonstrate that virus dose positively correlates with antiviral NK cell activity and function, which are at least partly driven by IL-15 and IL-18. Our results suggest that NK cell activity may be therapeutically enhanced by administering IL-15 and IL-18 in virus infections that inadequately activate NK cells.IMPORTANCE In infections with retroviruses, like HIV and FV infection of mice, NK cells clearly mediate antiviral activities, but they are usually not sufficient to prevent severe pathology. Here we show that the initial infection dose impacts the induction of an antiviral NK cell response during an acute retroviral infection, which had not investigated before. High-dose infection resulted in a strong NK cell functionality, whereas no antiviral activities were detected after low- or medium-dose infection. Interestingly, DCs and macrophages were highly activated after high-dose FV challenge, which corresponded with increased levels of NK cell-stimulating cytokines IL-15 and IL-18. IL-15 and IL-18 neutralization decreased NK cell functions, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we show the importance of cytokines for NK cell activation in retroviral infections; our findings suggest that immunotherapy combining the well-tolerated cytokines IL-15 and IL-18 might be an interesting approach for antiretroviral treatment.

B Cell Requirement for Robust Regulatory T Cell Responses to Friend Retrovirus Infection.

Regulatory T cells (Tregs) are immunosuppressive cells of the immune system that control autoimmune reactivity. Tregs also respond during immune reactions to infectious agents in order to limit immunopathological damage from potent effectors such as CD8+ cytolytic T lymphocytes. We have used the Friend virus (FV) model of retroviral infection in mice to investigate how viral infections induce Tregs. During acute FV infection, there is significant activation and expansion of thymus-derived (natural) Tregs that suppress virus-specific CD8+ T cell responses. Unlike conventional T cells, the responding Tregs are not virus specific, so the mechanisms that induce their expansion are of great interest. We now show that B cells provide essential signals for Treg expansion during FV infection. Treg responses are greatly diminished in B cell-deficient mice but can be restored by adoptive transfers of B cells at the time of infection. The feeble Treg responses in B cell-deficient mice are associated with enhanced virus-specific CD8+ T cell responses and accelerated virus control during the first 2 weeks of infection. In vitro experiments demonstrated that B cells promote Treg activation and proliferation through a glucocorticoid-induced receptor superfamily member 18 (GITR) ligand-dependent mechanism. Thus, B cells play paradoxically opposing roles during FV infection. They provide proliferative signals to immunsosuppressive Tregs, which slows early virus control, and they also produce virus-specific antibodies, which are essential for long-term virus control.IMPORTANCE When infectious agents invade a host, numerous immunological mechanisms are deployed to limit their replication, neutralize their spread, and destroy the host cells harboring the infection. Since immune responses also have a strong capacity to damage host cells and tissues, their magnitude, potency, and duration are under regulatory control. Regulatory T cells are an important component of this control, and the mechanisms that induce them to respond and exert immunosuppressive regulation are of great interest. In the current report, we show that B cells, the cells responsible for making pathogen-specific antibodies, are also involved in promoting the expansion of regulatory T cells during a retroviral infection. In vitro studies demonstrated that they do so via stimulation of the Tregs through interactions between cell surface molecules: GITR interactions with its ligand (GITRL) on B cells and GITR on regulatory T cells. These findings point the way toward therapeutics to better treat infections and autoimmune diseases.

Granulocytic myeloid-derived suppressor cells suppress virus-specific CD8+ T cell responses during acute Friend retrovirus infection.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) can suppress T cell responses in several different diseases. Previously these suppressive cells were observed to expand in HIV patients and in a mouse retrovirus model, yet their suppressive effect on virus-specific CD8+ T cells in vitro and in vivo has not been characterized thus far. RESULTS: We used the Friend retrovirus (FV) model to demonstrate that MDSCs expand and become activated during the late phase of acute FV infection. Only the subpopulation of granulocytic MDSCs (gMDSCs) but not monocytic MDSC suppressed virus-specific CD8+ T cell proliferation and function in vitro. gMDSCs expressed arginase 1, high levels of the inhibitory ligand PD-L1 and the ATP dephosphorylating enzyme CD39 on the cell surface upon infection. All three molecules were involved in the suppressive effect of the gMDSCs in vitro. MDSC depletion experiments in FV-infected mice revealed that they restrict virus-specific CD8+ T cell responses and thus affect the immune control of chronic retroviruses in vivo. CONCLUSIONS: Our study demonstrates that MDSCs become activated and expand during the acute phase of retrovirus infection. Their suppressive activity on virus-specific CD8+ T cells may contribute to T cell dysfunction and the development of chronic infection.

Fas Ligand-mediated cytotoxicity of CD4+ T cells during chronic retrovirus infection.

CD4+ helper T cells and cytotoxic CD8+ T cells are key players for adaptive immune responses against acute infections with retroviruses. Similar to textbook knowledge the most important function of CD4+ T cells during an acute retrovirus infection seems to be their helper function for other immune cells. Whereas there was no direct anti-viral activity of CD4+ T cells during acute Friend Virus (FV) infection, they were absolutely required for the control of chronic infection. During chronic FV infection a population of activated FV-specific CD4+ T cells did not express cytotoxic molecules, but Fas Ligand that can induce Fas-induced apoptosis in target cells. Using an MHC II-restricted in vivo CTL assay we demonstrated that FV-specific CD4+ T cells indeed mediated cytotoxic effects against FV epitope peptide loaded targets. CD4 + CTL killing was also detected in FV-infected granzyme B knockout mice confirming that the exocytosis pathway was not involved. However, killing could be blocked by antibodies against FasL, which identified the Fas/FasL pathway as critical cytotoxic mechanism during chronic FV infection. Interestingly, targeting the co-stimulatory receptor CD137 with an agonistic antibody enhanced CD4+ T cell cytotoxicity. This immunotherapy may be an interesting new approach for the treatment of chronic viral infections.

Immunodominance of Adenovirus-Derived CD8+ T Cell Epitopes Interferes with the Induction of Transgene-Specific Immunity in Adenovirus-Based Immunization.

Adenovirus (Ad)-based immunization is a popular approach in vaccine development, and Ad-based vectors are renowned for their potential to induce strong CD8+ T cell responses to the encoded transgene. Surprisingly, we previously found in the mouse Friend retrovirus (FV) model that Ad-based immunization did not induce CD8+ T cell responses to the FV Leader-Gag-derived immunodominant epitope GagL85-93 We show now that induction of GagL85-93-specific CD8+ T cells was highly effective when leader-Gag was delivered by plasmid DNA immunization, implying a role for Ad-derived epitopes in mediating unresponsiveness. By immunizing with DNA constructs encoding strings of GagL85-93 and the two Ad-derived epitopes DNA-binding protein418-426 (DBP418-426) and hexon486-494, we confirmed that Ad epitopes prevent induction of GagL85-93-specific CD8+ T cells. Interestingly, while DBP418-426 did not interfere with GagL85-93-specific CD8+ T cell induction, the H-2Dd-restricted hexon486-494 suppressed the CD8+ T cell response to the H-2Db-restricted GagL85-93 strongly in H-2b/d mice but not in H-2b/b mice. This finding indicates that competition occurs at the level of responding CD8+ T cells, and we could indeed demonstrate that coimmunization with an interleukin 2 (IL-2)-encoding plasmid restored GagL85-93-specific CD8+ T cell responses to epitope strings in the presence of hexon486-494 IL-2 codelivery did not restore GagL85-93 responsiveness in Ad-based immunization, however, likely due to the presence of further epitopes in the Ad vector. Our findings show that seemingly immunodominant transgene epitopes can be dominated by Ad-derived epitopes. These findings underline the importance of thorough characterization of vaccine vectors, and modifications of vectors or immunogens may be required to prevent impaired transgene-specific immune responses.IMPORTANCE Ad-based vectors are widely used in experimental preclinical and clinical immunization studies against numerous infectious agents, such as human immunodeficiency virus, Ebola virus, Plasmodium falciparum, or Mycobacterium tuberculosis Preexisting immunity to Ad-based vectors is widely recognized as a hindrance to the widespread use of Ad-based vectors for immunizations in humans; however, our data show that an immune response to Ad-derived T cell epitopes can also result in loss or impairment of transgene-specific immune responses in prenaive vaccinees due to immune competition. Our results highlight that seemingly immunodominant epitopes may be affected by dominance of vector-derived epitopes, and modifications of the vector design or the immunogens employed in immunization may lead to more effective vaccines.

Interference of retroviral envelope with vaccine-induced CD8+ T cell responses is relieved by co-administration of cytokine-encoding vectors.

BACKGROUND: Retroviral envelope (Env) proteins are known to exhibit immunosuppressive properties, which become apparent not only in retroviral infections, but also in gene-based immunizations using retroviral immunogens, where envelope interferes with the induction of CD8+ T cell responses towards another, simultaneously or subsequently delivered, immunogen. RESULTS: In the Friend retrovirus mouse model, immunization with a plasmid encoding the Friend murine leukemia virus (F-MuLV) Leader-Gag protein resulted in induction of a strong GagL85-93-specific CD8+ T cell response, while the response was completely abrogated by co-immunization with an F-MuLV Env-encoding plasmid. In order to overcome this interference of retroviral envelope, we employed plasmids encoding the cytokines interleukin (IL) 1ß, IL2, IL12, IL15, IL21, IL28A or granulocyte-macrophage colony-stimulating factor (GM-CSF) as genetic adjuvants. Co-application of plasmids encoding IL2, IL12, IL21, IL28A and especially GM-CSF rescued the induction of GagL85-93-specific CD8+ T cells in mice vaccinated with FV Leader-Gag and Env. Mice that were immunized with plasmids encoding Leader-Gag and Env and the cytokines IL1ß, IL12, IL15, IL28A or GM-CSF, but not Leader-Gag and Env without any cytokine, showed significantly reduced viral loads upon a high-dose Friend virus challenge infection. CONCLUSIONS: Our data demonstrate the potency of cytokine-encoding vectors as adjuvants and immune modulators in composite vaccines for anti-retroviral immunization.

Type I interferon signaling is required for the APOBEC3/Rfv3-dependent neutralizing antibody response but not innate retrovirus restriction.

BACKGROUND: APOBEC3/Rfv3 restricts acute Friend retrovirus (FV) infection and promotes virus-specific neutralizing antibody (NAb) responses. Classical Rfv3 studies utilized FV stocks containing lactate-dehydrogenase elevating virus (LDV), a potent type I interferon inducer. Previously, we showed that APOBEC3 is required for the anti-FV activity of exogenous IFN-alpha treatment. Thus, type I interferon receptor (IFNAR) signaling may be required for the APOBEC3/Rfv3 response. RESULTS: To test if the APOBEC3/Rfv3 response is dependent on type I IFN signaling, we infected IFNAR knockout versus IFNAR/APOBEC3 double-knockout mice with FV/LDV or LDV-free FV, and evaluated acute FV infection and subsequent NAb titers. We show that LDV co-infection and type I IFN signaling are not required for innate APOBEC3-mediated restriction. By contrast, removal of LDV and/or type I IFN signaling abrogated the APOBEC3-dependent NAb response. CONCLUSIONS: APOBEC3 can restrict retroviruses in a type I IFN-independent manner in vivo. By contrast, the ability of APOBEC3 to promote NAb responses is type I IFN-dependent. These findings reveal novel insights on the interplay between type I IFNs and APOBEC3 in vivo that may have implications for augmenting antiretroviral NAb responses.