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With increasing numbers of bispecific antibodies (BsAbs) and multispecific products entering the clinic, recent data highlight immunogenicity as an emerging challenge in the development of such novel biologics. This review focuses on the immunogenicity risk assessment (IgRA) of BsAb-based immunotherapies for cancer, highlighting several risk factors that need to be considered. These include the novel scaffolds consisting of bioengineered sequences, the potentially synergistic immunomodulating mechanisms of action (MOAs) from different domains of the BsAb, as well as several other product-related and patient-related factors. In addition, the clinical relevance of anti-drug antibodies (ADAs) against selected BsAbs developed as anticancer agents is reviewed and the advances in our knowledge of tools and strategies for immunogenicity prediction, monitoring, and mitigation are discussed. It is critical to implement a drug-specific IgRA during the early development stage to guide ADA monitoring and risk management strategies. This IgRA may include a combination of several assessment tools to identify drug-specific risks as well as a proactive risk mitigation approach for candidate or format selection during the preclinical stage. The IgRA is an on-going process throughout clinical development. IgRA during the clinical stage may bridge the gap between preclinical immunogenicity prediction and clinical immunogenicity, and retrospectively guide optimization efforts for next-generation BsAbs. This iterative process throughout development may improve the reliability of the IgRA and enable the implementation of effective risk mitigation strategies, laying the foundation for improved clinical success.

Despite 30 years of research on HIV, a vaccine to prevent infection and limit disease progression remains elusive. The RV144 trial showed moderate, but significant protection in humans and highlighted the contribution of antibody responses directed against HIV envelope as an important immune correlate for protection. Efforts to further build upon the progress include the use of a heterologous prime-boost regimen using DNA as the priming agent and the attenuated vaccinia virus, Modified Vaccinia Ankara (MVA), as a boosting vector for generating protective HIV-specific immunity. Areas covered: In this review, we summarize the immunogenicity of DNA/MVA vaccines in non-human primate models and describe the efficacy seen in SIV infection models. We discuss immunological correlates of protection determined by these studies and potential approaches for improving the protective immunity. Additionally, we describe the current progress of DNA/MVA vaccines in human trials. Expert commentary: Efforts over the past decade have provided the opportunity to better understand the dynamics of vaccine-induced immune responses and immune correlates of protection against HIV. Based on what we have learned, we outline multiple areas where the field will likely focus on in the next five years.

Activated CD4 T cells are a major target of HIV infection. Results from the STEP HIV vaccine trial highlighted a potential role for total activated CD4 T cells in promoting HIV acquisition. However, the influence of vaccine insert-specific CD4 T cell responses on HIV acquisition is not known. Here, using the data obtained from four macaque studies, we show that the DNA prime/modified vaccinia Ankara boost vaccine induced interferon γ (IFNγ+) CD4 T cells [T helper 1 (TH1) cells] rapidly migrate to multiple tissues including colon, cervix, and vaginal mucosa. These mucosal TH1 cells persisted at higher frequencies and expressed higher density of CCR5, a viral coreceptor, compared to cells in blood. After intravaginal or intrarectal simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV) challenges, strong vaccine protection was evident only in animals that had lower frequencies of vaccine-specific TH1 cells but not in animals that had higher frequencies of TH1 cells, despite comparable vaccine-induced humoral and CD8 T cell immunity in both groups. An RNA transcriptome signature in blood at 7 days after priming immunization from one study was associated with induction of fewer TH1-type CD4 cells and enhanced protection. These results demonstrate that high and persisting frequencies of HIV vaccine-induced TH1-biased CD4 T cells in the intestinal and genital mucosa can mitigate beneficial effects of protective antibodies and CD8 T cells, highlighting a critical role of priming immunization and vaccine adjuvants in modulating HIV vaccine efficacy.

We tested, in rhesus macaques, the effects of a 500-fold range of an admixed recombinant modified vaccinia Ankara (MVA) expressing rhesus GM-CSF (MVA/GM-CSF) on the immunogenicity and protection elicited by an MVA/SIV macaque 239 vaccine. High doses of MVA/GM-CSF did not affect the levels of systemic envelope (Env)-specific Ab, but it did decrease the expression of the gut-homing receptor α4β7 on plasmacytoid dendritic cells (p < 0.01) and the magnitudes of Env-specific IgA (p = 0.01) and IgG (p < 0.05) in rectal secretions. The protective effect of the vaccine was evaluated using 12 weekly rectal challenges in rhesus macaques subgrouped by tripartite motif-containing protein 5α (TRIM5α) genotypes that are restrictive or permissive for infection by the challenge virus SIVsmE660. Eight of nine TRIM5α-restrictive animals receiving no or the lowest dose (1 × 105 PFU) of MVA/GM-CSF resisted all 12 challenges. In the comparable TRIM5α-permissive group, only 1 of 12 animals resisted all 12 challenges. In the TRIM5α-restrictive animals, but not in the TRIM5α-permissive animals, the number of challenges to infection directly correlated with the magnitudes of Env-specific rectal IgG (r = +0.6) and IgA (r = +0.6), the avidity of Env-specific serum IgG (r = +0.5), and Ab dependent cell-mediated virus inhibition (r = +0.6). Titers of neutralizing Ab did not correlate with protection. We conclude that 1) protection elicited by MVA/SIVmac239 is strongly dependent on the presence of TRIM5α restriction, 2) nonneutralizing Ab responses contribute to protection against SIVsmE660 in TRIM5α-restrictive animals, and 3) high doses of codelivered MVA/GM-CSF inhibit mucosal Ab responses and the protection elicited by MVA expressing noninfectious SIV macaque 239 virus-like particles.

Modified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13R by replacing the fragmented 181R/182R genes of MVA with a functional anti-apoptotic gene, B13R, and confirmed its anti-apoptotic function against chemically induced apoptosis in vitro In addition, MVA-B13R showed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141+ DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13R expressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cells in vitro Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoring B13R functionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cells in vitro and augments vaccine-induced humoral immunity in mice.IMPORTANCE MVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes 181R/182R with a functional version derived from vaccinia virus, B13R Our results show that MVA-B13R significantly delays apoptosis in antigen-presenting cells and muscle cells in vitro and augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

Therapeutic strategies that augment antiviral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The coinhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of antiviral CD8+ T cells and B cells. Here, we tested the immunological and virological effects of PD-1 blockade combined with antiretroviral therapy (ART) in rhesus macaques. Administration of anti–PD-1 antibody 10 days prior to ART initiation rapidly enhanced antiviral CD8+ T cell function and diminished IFN-stimulated genes. This resulted in faster viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. PD-1 blockade during ART resulted in lower levels of cell-associated replication-competent virus. Following ART interruption, PD-1 antibody–treated animals showed markedly higher expansion of proliferating CXCR5+perforin+granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in better control of viremia. Our results show that PD-1 blockade can be administered safely with ART to augment antiviral CD8+ T cell function and reduce the viral reservoir, leading to improved control of viral rebound after ART interruption.

Modified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13R by replacing the fragmented 181R/182R genes of MVA with a functional anti-apoptotic gene, B13R, and confirmed its anti-apoptotic function against chemically induced apoptosis in vitro. In addition, MVA-B13R showed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141(+) DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13R expressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cells in vitro. Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoring B13R functionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cells in vitro and augments vaccine-induced humoral immunity in mice. IMPORTANCE MVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes 181R/182R with a functional version derived from vaccinia virus, B13R. Our results show that MVA-B13R significantly delays apoptosis in antigen-presenting cells and muscle cells in vitro and augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

Liver eosinophilia has been associated with incidences of drug-induced liver injury (DILI) for more than 50 years, although its role in this disease has remained largely unknown. In this regard, it was recently shown that eosinophils played a pathogenic role in a mouse model of halothane-induced liver injury (HILI). However, the signaling events that drove hepatic expression of eosinophil-associated chemokines, eotaxins, eosinophil infiltration, and subsequent HILI were unclear. We now provide evidence implicating hepatic epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) and type 2 immunity, in particular, interleukin-4 (IL-4) production, in mediating hepatic eosinophilia and injury during HILI. TSLP was constitutively expressed by mouse hepatocytes and increased during HILI. Moreover, the severity of HILI was reduced in mice deficient in either the TSLP receptor (TSLPR) or IL-4 and was accompanied by decreases in serum levels of eotaxins and hepatic eosinophilia. Similarly, concanavalin A–induced liver injury, where type 2 cytokines and eosinophils play a significant role in its pathogenesis, was also reduced in TSLPR-deficient mice. Studies in vitro revealed that mouse and human hepatocytes produce TSLP and eotaxins in response to treatment with combinations of IL-4 and proinflammatory cytokines IL-1β and tumor necrosis factor alpha. Conclusion: This report provides the first evidence implicating roles for hepatic TSLP signaling, type 2 immunity, and eosinophilia in mediating liver injury caused by a drug. (Hepatology 2014;60:1741-1752)

Drug-induced liver injury (DILI) is a major health issue, as it remains difficult to predict which new drugs will cause injury and who will be susceptible to this disease. This is due in part to the lack of animal models and knowledge of susceptibility factors that predispose individuals to DILI. In this regard, liver eosinophilia has often been associated with DILI, although its role remains unclear. We decided to investigate this problem in a murine model of halothane-induced liver injury (HILI). When female Balb/cJ mice were administered halothane, eosinophils were detected by flow cytometry in the liver within 12 hours and increased thereafter proportionally to liver damage. Chemokines, eotaxin-1 (CCL11) and eotaxin-2 (CCL24), which are known to attract eosinophils, increased in response to halothane treatment. The severity of HILI was decreased significantly when the study was repeated in wildtype mice made deficient in eosinophils with a depleting antibody and in eosinophil lineage-ablated ΔdblGata−/− mice. Moreover, depletion of neutrophils by pretreating animals with Gr-1 antibody prior to halothane administration failed to reduce the severity of HILI at antibody concentrations that did not affect hepatic eosinophils. Immunohistochemical staining for the granule protein, major basic protein, revealed that eosinophils accumulated exclusively around areas of hepatocellular necrosis. Conclusion: Our findings indicate that eosinophils have a pathologic role in HILI in mice and suggest that they may contribute similarly in many clinical cases of DILI. (HEPATOLOGY 2013)