Zika virus (ZIKV) infection during human pregnancy may cause diverse and serious congenital defects in the developing fetus. Previous efforts to generate animal models of human ZIKV infection and clinical symptoms often involved manipulating mice to impair their Type I interferon (IFN) signaling, thereby allowing enhanced infection and vertical transmission of virus to the embryo. Here, we show that even pregnant mice competent to generate Type I IFN responses that can limit ZIKV infection nonetheless develop profound placental pathology and high frequency of fetal demise. We consistently found that maternal ZIKV exposure led to placental pathology and that ZIKV RNA levels measured in maternal, placental or embryonic tissues were not predictive of the pathological effects seen in the embryos. Placental pathology included trophoblast hyperplasia in the labyrinth, trophoblast giant cell necrosis in the junctional zone, and loss of embryonic vessels. Our findings suggest that, in this context of limited infection, placental pathology rather than embryonic/fetal viral infection may be a stronger contributor to adverse pregnancy outcomes in mice. Our finding demonstrates that in immunocompetent mice, direct viral infection of the embryo is not essential for fetal demise. Our immunologically unmanipulated pregnancy mouse model provides a consistent and easily measurable congenital abnormality readout to assess fetal outcome, and may serve as an additional model to test prophylactic and therapeutic interventions to protect the fetus during pregnancy, and for studying the mechanisms of ZIKV congenital immunopathogenesis. Zika virus (ZIKV) infection during human pregnancy may cause severe congenital abnormalities and fetal death. There is currently no licensed vaccine or anti-ZIKV therapeutic to prevent or treat infection and/or disease. To generate an animal model that mimics human ZIKV infections, others have manipulated mice to impair their innate immunity, which allows ZIKV to develop high levels of infection. Here, we present an experimental model using immunologically unmanipulated pregnant mice and show that even limited maternal ZIKV infection nonetheless resulted in profound placental pathology and high frequency of fetal demise. However, neither viral RNA level in the dam, placenta or embryo reliably predicted fetal abnormalities. Our studies suggest that, in this model, placental pathology including trophoblast hyperplasia, focal regions of necrosis, and loss of embryonic blood vessels in the placenta likely promote adverse fetal outcomes. This immunocompetent pregnant mouse model provides clear in vivo phenotypic readouts (e.g. embryonic viability and gross uterine and embryonic morphology) to assess the potential for clinical benefit of candidate vaccines and therapeutics in a model not dependent on vertical transmission of virus.

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE_69–77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE_69–77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE_69–77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE_69–77-mediated protection. In contrast, YopE_69–77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias. Bacterial pneumonia is one of the most common causes of death worldwide. Pulmonary infection of bacterium Yersinia pestis, the causative agent of plague, results in pneumonic plague and is extremely lethal. Mouse models of pulmonary Y. pestis infection are considered translational tools for the development of pneumonic plague countermeasures and studies of the basic mechanisms of immune defense against acutely lethal pulmonary bacterial infections. Here, we used several methods to investigate the functions that CD8 T cells exert to confer protection against pulmonary Y. pestis infection and evaluated their relative contributions. We found that although CD8 T cells have the ability to kill Y. pestis-infected cells, a function called cytotoxicity, this function is not required for CD8 T cells to protect against Y. pestis infection. In contrast, protection depends upon the ability of CD8 T cells to produce the cytokines TNFα and IFNγ, and mice whose T cells cannot produce these two cytokines are not protected. Therefore, we conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting cytokine production may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.

Immune checkpoint inhibitor-based combination therapy has demonstrated high objective response rates in patients with hepatocellular carcinoma (HCC) and may improve clinical outcomes as neoadjuvant or perioperative therapy. This study evaluated the efficacy and safety of nivolumab plus ipilimumab in patients with potentially resectable HCC and explored predictive biomarkers of treatment response. Eligible patients received nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg) every 3 weeks. Tumor response was assessed after two and four treatment cycles. Patients then proceeded to curative surgery or alternative treatments according to clinical guidelines. Serial tumor and peripheral blood samples were collected for genomic and transcriptomic analyses and immune cell profiling. Among the 43 enrolled patients (men/women: 37/6; viral/non-viral: 41/2; Barcelona Clinic Liver Cancer stage A/B/C: 4/13/26; median tumor size: 8.7 cm), 24 underwent surgery, and eight achieved a major pathological response (>90% tumor necrosis). The estimated 4-year progression-free survival and overall survival rates were 44% (95% CI 28-59%) and 60% (95% CI 42-74%), respectively. Increased interferon-γ and tertiary lymphoid structure (TLS) signatures in resected tumors were associated with objective response. In a mouse liver cancer model, B-cell depletion abolished the efficacy of anti-programmed death-1 plus anti-cytotoxic T lymphocyte-associated protein 4 therapy, supporting the mechanistic role of TLS. Peripheral blood T-cell activation and exhaustion at baseline and post-immunotherapy, assessed using spectral flow cytometry and deep learning algorithms, correlated with response and survival. Neoadjuvant nivolumab plus ipilimumab followed by surgery is feasible and may improve long-term survival in patients with potentially resectable HCC. Immunotherapy-induced TLS formation is associated with enhanced antitumor immunity. Immune checkpoint inhibitor-based systemic therapy is now an integral component of the multidisciplinary management of patients with hepatocellular carcinoma. Findings from this single-arm clinical trial indicate that: i) neoadjuvant nivolumab plus ipilimumab followed by surgery is feasible and may improve long-term survival in patients with potentially resectable hepatocellular carcinoma; ii) immunotherapy-induced tertiary lymphoid structure formation is associated with enhanced antitumor immunity; iii) monitoring T-cell exhaustion in peripheral blood may aid in the prediction of response to immunotherapy. NCT03510871.

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combined with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.

Zika virus (ZIKV) infection during pregnancy can result in a variety of developmental abnormalities in the fetus, referred to as Congenital Zika Syndrome (CZS). The effects of CZS can range from the loss of the viable fetus to a variety of neurological defects in full-term infants, including microcephaly. The clinical importance of ZIKV-induced CZS has driven an intense effort to develop effective vaccines. Consequently, there are approximately 45 different ZIKV vaccine candidates at various stages of development with several undergoing phase I and II clinical trials. These vaccine candidates have been shown to effectively prevent infection in adult animal models, however, there has been less extensive testing for their ability to block vertical transmission to the fetus during pregnancy or prevent the development of CZS. In addition, it is becoming increasingly difficult to test vaccines in the field as the intensity of the ZIKV epidemic has declined precipitously, making clinical endpoint studies difficult. These ethical and practical challenges in determining efficacy of ZIKV vaccine candidates in preventing CZS have led to increased emphasis on pre-clinical testing in animal pregnancy models. Here we review the current status of pre-clinical pregnancy models for testing the ability of ZIKV vaccines to prevent CZS.