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The causal association of Zika virus (ZIKV) with microcephaly, congenital malformations in infants, and Guillain-Barré syndrome in adults highlights the need for effective vaccines. Thus far, efforts to develop ZIKV vaccines have focused on the viral envelope. ZIKV NS1 as a vaccine immunogen has not been fully explored, although it can circumvent the risk of antibody-dependent enhancement of ZIKV infection, associated with envelope antibodies. Here, we describe a novel DNA vaccine encoding a secreted ZIKV NS1, that confers rapid protection from systemic ZIKV infection in immunocompetent mice. We identify novel NS1 T cell epitopes in vivo and show that functional NS1-specific T cell responses are critical for protection against ZIKV infection. We demonstrate that vaccine-induced anti-NS1 antibodies fail to confer protection in the absence of a functional T cell response. This highlights the importance of using NS1 as a target for T cell-based ZIKV vaccines.

As the first responder to immunological challenges, the innate immune system shapes and regulates the ensuing adaptive immune response. Many clinical studies evaluating the role of innate immunity in initiating vaccine-elicited adaptive immune responses have largely been confined to blood due to inherent difficulty in acquiring tissue samples. However, the absence of vaccine-site and draining lymph node information limits understanding of early events induced by vaccination that could potentially shape vaccine-elicited immunity. We therefore utilized a mouse model to investigate the spatiotemporal evolution of the immune response within the first 24 hours following intramuscular adenovirus serotype 26 (Ad26) vector vaccination in tissues. We show that the Ad26 vaccine-elicited innate immune response commences by one hour and rapidly evolves in tissues and blood within the first 24 hours as reflected by the detection of cytokines, chemokines, cellular responses, and transcriptomic pathways. Furthermore, serum levels of IL-6, MIG, MIP-1α, and MIP-1β at 6 hours post-vaccination correlated with the frequency of vaccine-elicited memory CD8 + T cell responses evaluated at 60 days post-vaccination in blood and tissues. Taken together, our data suggests that the immune response to Ad26 vector vaccination commences quickly in tissues by one hour and that events by as early as 6 hours post-vaccination can shape vaccine-elicited CD8 + T cell responses at later memory time points.

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development., Competing Interests: Declaration of interests S.J.K., V.D., G.D., K.M., N.M., D.H.B., M.G.J., R.S.S., and R.G.J. are named inventors on a PCT patent application WO 2019/209974 describing ZIKV neutralizing antibodies and their use. D.H.B. has received grants from Novavax and personal fees from IGM Biosciences. M.H., A.A., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is also a shareholder of the company., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The emergence of the SARS-CoV-2 Omicron sublineages resulted in increased transmission rates and reduced protection from vaccines. To counteract these effects, multiple booster strategies were used in different countries, although data comparing their efficiency in improving protective immunity remain sparse, especially among vulnerable populations, including older adults. The inactivated CoronaVac vaccine was among the most widely distributed vaccine worldwide and was essential in the early control of SARS-CoV-2-related hospitalizations and deaths. However, it is not well understood whether homologous versus heterologous booster doses in those fully vaccinated with CoronaVac induce distinct humoral responses or whether these responses vary across age groups. We analyzed plasma antibody responses from CoronaVac-vaccinated younger or older individuals who received a homologous CoronaVac or heterologous BNT162b2 or ChAdOx1 booster vaccine. All three evaluated boosters resulted in increased virus-specific IgG titers 28 days after the booster dose. However, we found that both IgG titers against SARS-CoV-2 Spike or RBD and neutralization titers against Omicron sublineages were substantially reduced in participants who received homologous CoronaVac compared with the heterologous BNT162b2 or ChAdOx1 booster. This effect was specifically prominent in recipients >50 years of age. In this group, the CoronaVac booster induced low virus-specific IgG titers and failed to elevate neutralization titers against any Omicron sublineage. Our results point to the notable inefficiency of CoronaVac immunization and boosting in mounting protective antiviral humoral immunity, particularly among older adults, during the Omicron wave. These observations also point to benefits of heterologous regimens in high-risk populations fully vaccinated with CoronaVac.

Pre-existing immunity to flaviviruses can influence the outcome of subsequent flavivirus infections. Therefore, it is critical to determine whether baseline DENV immunity may influence subsequent ZIKV infection and the protective efficacy of ZIKV vaccines. In this study, we investigated the impact of pre-existing DENV immunity induced by vaccination on ZIKV infection and the protective efficacy of an inactivated ZIKV vaccine. Rhesus macaques and mice inoculated with a live attenuated DENV vaccine developed neutralizing antibodies (NAbs) to multiple DENV serotypes but no cross-reactive NAbs responses to ZIKV. Animals with baseline DENV NAbs did not exhibit enhanced ZIKV infection and showed no overall reduction in ZIKV vaccine protection. Moreover, passive transfer of purified DENV-specific IgG from convalescent human donors did not augment ZIKV infection in STAT2 -/- and BALB/c mice. In summary, these results suggest that baseline DENV immunity induced by vaccination does not significantly enhance ZIKV infection or impair the protective efficacy of candidate ZIKV vaccines in these models. These data can help inform immunization strategies in regions of the world with multiple circulating pathogenic flaviviruses., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: P.A., R.A.L., D.H.B., R.A.D., and K.H.E. are co-inventors on pending patent applications related to ZIKV vaccines.

Background: Zika virus (ZIKV) may cause severe congenital disease after maternal-fetal transmission. No vaccine is currently available., Objective: To assess the safety and immunogenicity of Ad26.ZIKV.001, a prophylactic ZIKV vaccine candidate., Design: Phase 1 randomized, double-blind, placebo-controlled clinical study. (ClinicalTrials.gov: NCT03356561)., Setting: United States., Participants: 100 healthy adult volunteers., Intervention: Ad26.ZIKV.001, an adenovirus serotype 26 vector encoding ZIKV M-Env, administered in 1- or 2-dose regimens of 5 × 10 10 or 1 × 10 11 viral particles (vp), or placebo., Measurements: Local and systemic adverse events; neutralization titers by microneutralization assay (MN50) and T-cell responses by interferon-γ enzyme-linked immunospot and intracellular cytokine staining; and protectivity of vaccine-induced antibodies in a subset of participants through transfer in an exploratory mouse ZIKV challenge model., Results: All regimens were well tolerated, with no safety concerns identified. In both 2-dose regimens, ZIKV neutralizing titers peaked 14 days after the second vaccination, with geometric mean MN50 titers (GMTs) of 1065.6 (95% CI, 494.9 to 2294.5) for 5 × 10 10 vp and 956.6 (595.8 to 1535.8) for 1 × 10 11 vp. Titers persisted for at least 1 year at a GMT of 68.7 (CI, 26.4-178.9) for 5 × 10 10 vp and 87.0 (CI, 29.3 to 258.6) for 1 × 10 11 vp. A 1-dose regimen of 1 × 10 11 vp Ad26.ZIKV.001 induced seroconversion in all participants 56 days after the first vaccination (GMT, 103.4 [CI, 52.7 to 202.9]), with titers persisting for at least 1 year (GMT, 90.2 [CI, 38.4 to 212.2]). Env-specific cellular responses were induced. Protection against ZIKV challenge was observed after antibody transfer from participants into mice, and MN50 titers correlated with protection in this model., Limitation: The study was conducted in a nonendemic area, so it did not assess safety and immunogenicity in a flavivirus-exposed population., Conclusion: The safety and immunogenicity profile makes Ad26.ZIKV.001 a promising candidate for further development if the need reemerges., Primary Funding Source: Janssen Vaccines and Infectious Diseases.

Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis, is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c (ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4+ T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4+ T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy., Competing Interests: The authors have declared that no competing interests exist. A.J.M. and D.H.B. are co-inventors on a TB vaccine patent.

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV.

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. 1-3 ). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV) 4-7 in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.

Adenoviral vectors have shown significant promise as vaccine delivery vectors due to their ability to elicit both innate and adaptive immune responses. α-defensins are effector molecules of the innate immune response and have been shown to modulate natural infection with adenoviruses, but the majority of α-defensin-adenovirus interactions studied to date have only been analyzed in vitro. In this study, we evaluated the role of α-defensin 5 (HD5) in modulating adenovirus vaccine immunogenicity using various serotype adenovirus vectors in mice. We screened a panel of human adenoviruses including Ad5 (species C), Ad26 (species D), Ad35 (species B), Ad48 (species D) and a chimeric Ad5HVR48 for HD5 sensitivity. HD5 inhibited transgene expression from Ad5 and Ad35 but augmented transgene expression from Ad26, Ad48, and Ad5HVR48. HD5 similarly suppressed antigen-specific IgG and CD8+ T cell responses elicited by Ad5 vectors in mice, but augmented IgG and CD8+ T cell responses and innate cytokine responses elicited by Ad26 vectors in mice. Moreover, HD5 suppressed the protective efficacy of Ad5 vectors but enhanced the protective efficacy of Ad26 vectors expressing SIINFEKL against a surrogate Listeria-OVA challenge in mice. These data demonstrate that HD5 differentially modulates adenovirus vaccine delivery vectors in a species-specific manner in vivo., Competing Interests: The authors have declared that no competing interests exist.

Maternal infection with Zika virus (ZIKV) can lead to microcephaly and other congenital abnormalities of the fetus. Although ZIKV vaccines that prevent or reduce viremia in non-pregnant mice have been described, a maternal vaccine that provides complete fetal protection would be desirable. Here, we show that adenovirus (Ad) vector-based ZIKV vaccines induce potent neutralizing antibodies that confer robust maternal and fetal protection against ZIKV challenge in pregnant, highly susceptible IFN-αβR -/- mice. Moreover, passive transfer of maternal antibodies from vaccinated dams protected pups against post-natal ZIKV challenge. These data suggest that Ad-based ZIKV vaccines may be able to provide protection in pregnant females against fetal ZIKV transmission in utero as well as in infants against ZIKV infection after birth., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has spread to more than 70 countries worldwide since 2015. Despite active research, there are currently no licensed vaccines or therapeutics. We have previously reported the development of various adenoviral vectored vaccine candidates (ChAdOx1 ZIKV) with the ability to stimulate effective immunity in mice and provide protection upon a ZIKV challenge model, using a non-adjuvanted single vaccination approach. In this study, we constructed various modified vaccinia Ankara (MVA) viruses to express the ZIKV Envelope (E) with modifications on the precursor membrane (prM) or on the C-terminus envelope transmembrane domain (TM), similar to our ChAdOx1 vaccine candidates. MVA-ZIKV vaccine candidates were evaluated as a non-adjuvanted single vaccination regimen against a ZIKV Brazilian isolate, using viraemia as the correlate of protection. Here, we report the induction of a modest level of anti-ZIKV E antibodies by all MVA vectored vaccines and sub-optimal efficacy in a ZIKV challenge model. Our results indicate the requirement of additional strategies when using MVA-ZIKV vaccines to afford sterile protection upon a non-adjuvanted and single vaccination regime., Competing Interests: A.R.-S. and C.L.C. are co-inventors of the Zika vaccines described in this manuscript, filed by Oxford University Innovation Limited in the International Patent Application No. PCR/GB2017/052220 Zika Vaccine. The remaining authors declare no competing interests.

The combined inhibition of histone deacetylases (HDAC) and the proteins of the bromodomain and extraterminal (BET) family have recently shown therapeutic efficacy against melanoma, pancreatic ductal adenocarcinoma, testicular, and lymphoma cancers in murine studies. However, in such studies, the role of the immune system in therapeutically controlling these cancers has not been explored. We sought to investigate the effect of the HDAC inhibitor romidepsin (RMD) and the BET inhibitor IBET151, both singly and in combination, on vaccine-elicited immune responses. C57BL/6 mice were immunized with differing vaccine systems (adenoviral, protein) in prime-boost regimens under treatment with RMD, IBET151, or RMD+IBET151. The combined administration of RMD+IBET151 during vaccination resulted in a significant increase in the frequency and number of Ag-specific CD8 + T cells. RMD+IBET151 treatment significantly increased the frequency of vaccine-elicited IFN-γ + splenic CD8 + T cells and conferred superior therapeutic and prophylactic protection against B16-OVA melanoma. RNA sequencing analyses revealed strong transcriptional similarity between RMD+IBET151 and untreated Ag-specific CD8 + T cells except in apoptosis and IL-6 signaling-related genes that were differentially expressed. Serum IL-6 was significantly increased in vivo following RMD+IBET151 treatment, with recombinant IL-6 administration replicating the effect of RMD+IBET151 treatment on vaccine-elicited CD8 + T cell responses. IL-6 sufficiency for protection was not assessed. Combined HDAC and BET inhibition resulted in greater vaccine-elicited CD8 + T cell responses and enhanced therapeutic and prophylactic protection against B16-OVA melanoma. Increased IL-6 production and the differential expression of pro- and anti-apoptotic genes following RMD+IBET151 treatment are likely contributors to the enhanced cancer vaccine responses., (Copyright © 2018 by The American Association of Immunologists, Inc.)

In 2015, there was a large outbreak of Zika virus (ZIKV) in Brazil. Despite its relatively mild impact on healthy adults, ZIKV infection during pregnancy has been associated with severe birth defects. Currently, there is no ZIKV vaccine available, but several vaccine candidates based on the ZIKV membrane (M) and envelope (Env) structural proteins showed promising results in preclinical and clinical studies. Here, the immunogenicity and protective efficacy of a non-replicating adenoviral vector type 26 (Ad26) that encodes the ZIKV M-Env antigens (Ad26.ZIKV.M-Env) was evaluated in mice and non-human primates (NHP). Ad26.ZIKV.M-Env induced strong and durable cellular and humoral immune responses in preclinical models. Humoral responses were characterized by Env-binding and ZIKV neutralizing antibody responses while cellular responses were characterized by ZIKV reactive CD4+ and CD8+ T cells. Importantly, a single immunization with a very low dose of 4x107 vp of Ad26.ZIKV.M-Env protected mice from ZIKV challenge. In NHP, a single immunization with a typical human dose of 1x1011 vp of Ad26.ZIKV.M-Env also induced Env-binding and ZIKV neutralizing antibodies and Env and M specific cellular immune responses that associated with complete protection against viremia from ZIKV challenge as measured in plasma and other body fluids. Together these data provide the rationale to progress the Ad26.ZIKV.M-Env candidate vaccine to clinical testing., Competing Interests: Janssen Vaccines & Prevention B.V, The Netherlands (Janssen) or Beth Israel Deaconess Medical Center, USA (BIDMC) funded the study without receiving external funding. All authors are employees of Janssen Vaccines & Prevention B.V or Beth Israel Deaconess Medical Center. DB, PA AND RAL are inventors on patents (International patent application number: PCT/US2017/036900 Title: Compositions and methods for preventing and treating ZIKA virus infection) that have been licensed to Janssen Vaccines & Prevention B.V. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Zika virus (ZIKV) emerged on a global scale and no licensed vaccine ensures long-lasting anti-ZIKV immunity. Here we report the design and comparative evaluation of four replication-deficient chimpanzee adenoviral (ChAdOx1) ZIKV vaccine candidates comprising the addition or deletion of precursor membrane (prM) and envelope, with or without its transmembrane domain (TM). A single, non-adjuvanted vaccination of ChAdOx1 ZIKV vaccines elicits suitable levels of protective responses in mice challenged with ZIKV. ChAdOx1 prME ∆TM encoding prM and envelope without TM provides 100% protection, as well as long-lasting anti-envelope immune responses and no evidence of in vitro antibody-dependent enhancement to dengue virus. Deletion of prM and addition of TM reduces protective efficacy and yields lower anti-envelope responses. Our finding that immunity against ZIKV can be enhanced by modulating antigen membrane anchoring highlights important parameters in the design of viral vectored ZIKV vaccines to support further clinical assessments.

Strategies to treat Zika virus (ZIKV) infection in dengue virus (DENV)-endemic areas are urgently needed. Here we show that a DENV-specific antibody against the E-dimer epitope (EDE) potently cross-neutralizes ZIKV and provides robust therapeutic efficacy as well as prophylactic efficacy against ZIKV in rhesus monkeys. Viral escape was not detected, suggesting a relatively high bar to escape. These data demonstrate the potential for antibody-based therapy and prevention of ZIKV.

The development of interventions to prevent congenital Zika syndrome (CZS) has been limited by the lack of an established nonhuman primate model. Here we show that infection of female rhesus monkeys early in pregnancy with Zika virus (ZIKV) recapitulates many features of CZS in humans. We infected 9 pregnant monkeys with ZIKV, 6 early in pregnancy (weeks 6-7 of gestation) and 3 later in pregnancy (weeks 12-14 of gestation), and compared findings with uninfected controls. 100% (6 of 6) of monkeys infected early in pregnancy exhibited prolonged maternal viremia and fetal neuropathology, including fetal loss, smaller brain size, and histopathologic brain lesions, including microcalcifications, hemorrhage, necrosis, vasculitis, gliosis, and apoptosis of neuroprogenitor cells. High-resolution MRI demonstrated concordant lesions indicative of deep gray matter injury. We also observed spinal, ocular, and neuromuscular pathology. Our data show that vascular compromise and neuroprogenitor cell dysfunction are hallmarks of CZS pathogenesis, suggesting novel strategies to prevent and to treat this disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Natural killer (NK) cells respond rapidly as a first line of defense against infectious pathogens. In addition, NK cells may provide a "rheostat" function and have been shown to reduce the magnitude of antigen-specific T cell responses following infection to avoid immunopathology. However, it remains unknown whether NK cells similarly modulate vaccine-elicited T cell responses following virus challenge. We used the lymphocytic choriomeningitis virus (LCMV) clone 13 infection model to address whether NK cells regulate T cell responses in adenovirus vector-vaccinated mice following challenge. As expected, NK cell depletion in unvaccinated mice resulted in increased virus-specific CD4 + and CD8 + T cell responses and immunopathology following LCMV challenge. In contrast, NK cell depletion had minimal to no impact on antigen-specific T cell responses in mice that were vaccinated with an adenovirus serotype 5 (Ad5)-GP vector prior to LCMV challenge. Moreover, NK cell depletion in vaccinated mice prior to challenge did not result in immunopathology and did not compromise protective efficacy. These data suggest that adenovirus vaccine-elicited T cells may be less sensitive to NK cell rheostat regulation than T cells primed by LCMV infection. IMPORTANCE Recent data have shown that NK cell depletion leads to enhanced virus-elicited T cell responses that can result in severe immunopathology following LCMV infection in mice. In this study, we observed that NK cells exerted minimal to no impact on vaccine-elicited T cells following LCMV challenge, suggesting that adenovirus vaccine-elicited T cells may be less subject to NK cell regulation. These data contribute to our understanding of NK cell regulatory functions and T cell-based vaccines., (Copyright © 2018 Blass et al.)

Adenovirus (Ad) vectors are being investigated as vaccine candidates, but baseline antivector immunity exists in human populations to both human Ad (HuAd) and chimpanzee Ad (ChAd) vectors. In this study, we investigated the immunogenicity and cross-reactivity of a panel of recently described rhesus adenoviral (RhAd) vectors. RhAd vectors elicited T cells with low exhaustion markers and robust anamnestic potential. Moreover, RhAd vector immunogenicity was unaffected by high levels of preexisting anti-HuAd immunity. Both HuAd/RhAd and RhAd/RhAd prime-boost vaccine regimens were highly immunogenic, despite a degree of cross-reactive neutralizing antibodies (NAbs) between phylogenetically related RhAd vectors. We observed extensive vector-specific cross-reactive CD4 T cell responses and more limited CD8 T cell responses between RhAd and HuAd vectors, but the impact of vector-specific cellular responses was far less than that of vector-specific NAbs. These data suggest the potential utility of RhAd vectors and define novel heterologous prime-boost strategies for vaccine development. IMPORTANCE To date, most adenoviral vectors developed for vaccination have been HuAds from species B, C, D, and E, and human populations display moderate to high levels of preexisting immunity. There is a clinical need for new adenoviral vectors that are not hindered by preexisting immunity. Moreover, the development of RhAd vector vaccines expands our ability to vaccinate against multiple pathogens in a population that may have received other HuAd or ChAd vectors. We evaluated the immunogenicity and cross-reactivity of RhAd vectors, which belong to the poorly described adenovirus species G. These vectors induced robust cellular and humoral immune responses and were not hampered by preexisting anti-HuAd vector immunity. Such properties make RhAd vectors attractive as potential vaccine vectors., (Copyright © 2018 Iampietro et al.)

Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice., (Copyright © 2018 Badamchi-Zadeh et al.)

Human and chimpanzee adenovirus vectors are being developed to circumvent preexisting antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes 2 months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development. IMPORTANCE To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development., (Copyright © 2018 Abbink et al.)

Background: A safe, effective, and rapidly scalable vaccine against Zika virus infection is needed. We developed a purified formalin-inactivated Zika virus vaccine (ZPIV) candidate that showed protection in mice and non-human primates against viraemia after Zika virus challenge. Here we present the preliminary results in human beings., Methods: We did three phase 1, placebo-controlled, double-blind trials of ZPIV with aluminium hydroxide adjuvant. In all three studies, healthy adults were randomly assigned by a computer-generated list to receive 5 μg ZPIV or saline placebo, in a ratio of 4:1 at Walter Reed Army Institute of Research, Silver Spring, MD, USA, or of 5:1 at Saint Louis University, Saint Louis, MO, USA, and Beth Israel Deaconess Medical Center, Boston, MA, USA. Vaccinations were given intramuscularly on days 1 and 29. The primary objective was safety and immunogenicity of the ZPIV candidate. We recorded adverse events and Zika virus envelope microneutralisation titres up to day 57. These trials are registered at ClinicalTrials.gov, numbers NCT02963909, NCT02952833, and NCT02937233., Findings: We enrolled 68 participants between Nov 7, 2016, and Jan 25, 2017. One was excluded and 67 participants received two injections of Zika vaccine (n=55) or placebo (n=12). The vaccine caused only mild to moderate adverse events. The most frequent local effects were pain (n=40 [60%]) or tenderness (n=32 [47%]) at the injection site, and the most frequent systemic reactogenic events were fatigue (29 [43%]), headache (26 [39%]), and malaise (15 [22%]). By day 57, 52 (92%) of vaccine recipients had seroconverted (microneutralisation titre ≥1:10), with peak geometric mean titres seen at day 43 and exceeding protective thresholds seen in animal studies., Interpretation: The ZPIV candidate was well tolerated and elicited robust neutralising antibody titres in healthy adults., Funding: Departments of the Army and Defense and National Institute of Allergy and Infectious Diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

An effective Zika virus (ZIKV) vaccine will require long-term durable protection. Several ZIKV vaccine candidates have demonstrated protective efficacy in nonhuman primates, but these studies have typically involved ZIKV challenge shortly after vaccination at peak immunity. We show that a single immunization with an adenovirus vector-based vaccine, as well as two immunizations with a purified inactivated virus vaccine, afforded robust protection against ZIKV challenge in rhesus monkeys at 1 year after vaccination. In contrast, two immunizations with an optimized DNA vaccine, which provided complete protection at peak immunity, resulted in reduced protective efficacy at 1 year that was associated with declining neutralizing antibody titers to subprotective levels. These data define a microneutralization log titer of 2.0 to 2.1 as the threshold required for durable protection against ZIKV challenge in this model. Moreover, our findings demonstrate that protection against ZIKV challenge in rhesus monkeys is possible for at least 1 year with a single-shot vaccine., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Zika virus (ZIKV) is associated with severe neuropathology in neonates as well as Guillain-Barré syndrome and other neurologic disorders in adults. Prolonged viral shedding has been reported in semen, suggesting the presence of anatomic viral reservoirs. Here we show that ZIKV can persist in cerebrospinal fluid (CSF) and lymph nodes (LN) of infected rhesus monkeys for weeks after virus has been cleared from peripheral blood, urine, and mucosal secretions. ZIKV-specific neutralizing antibodies correlated with rapid clearance of virus in peripheral blood but remained undetectable in CSF for the duration of the study. Viral persistence in both CSF and LN correlated with upregulation of mechanistic target of rapamycin (mTOR), proinflammatory, and anti-apoptotic signaling pathways, as well as downregulation of extracellular matrix signaling pathways. These data raise the possibility that persistent or occult neurologic and lymphoid disease may occur following clearance of peripheral virus in ZIKV-infected individuals., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Adenovirus serotype 5 (Ad5) vaccine vectors elicit robust CD8 + T cell responses, but these responses typically exhibit a partially exhausted phenotype. However, the immunologic mechanism by which Ad5 vectors induce dysfunctional CD8 + T cells has not previously been elucidated. Here we demonstrate that, following immunization of B6 mice, Ad5 vectors elicit antigen-specific IL-10 + CD4 + T cells with a distinct transcriptional profile in a dose-dependent fashion. In rhesus monkeys, we similarly observed upregulated expression of IL-10 and PD-1 by CD4 + T cells following Ad5 vaccination. These cells markedly suppressed vaccine-elicited CD8 + T cell responses in vivo and IL-10 blockade increased the frequency and functionality of antigen-specific CD8 + T cells as well as improved protective efficacy against challenge with recombinant Listeria monocytogenes . Moreover, induction of these inhibitory IL-10 + CD4 + T cells correlated with IL-27 expression and IL-27 blockade substantially improved CD4 + T cell functionality. These data highlight a role for IL-27 in the induction of inhibitory IL-10 + CD4 + T cells, which suppress CD8 + T cell magnitude and function following Ad5 vector immunization. A deeper understanding of the cytokine networks and transcriptional profiles induced by vaccine vectors should lead to strategies to improve the immunogenicity and protective efficacy of viral vector-based vaccines., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys also conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector vaccine, both expressing ZIKV premembrane and envelope, also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans., (Copyright © 2016, American Association for the Advancement of Science.)

CD4(+) T cell help is critical for optimal CD8(+) T cell memory differentiation and maintenance in many experimental systems. In addition, many reports have identified reduced primary CD8(+) T cell responses in the absence of CD4(+) T cell help, which often coincides with reduced Ag or pathogen clearance. In this study, we demonstrate that absence of CD4(+) T cells at the time of adenovirus vector immunization of mice led to immediate impairments in early CD8(+) T cell functionality and differentiation. Unhelped CD8(+) T cells exhibited a reduced effector phenotype, decreased ex vivo cytotoxicity, and decreased capacity to produce cytokines. This dysfunctional state was imprinted within 3 d of immunization. Unhelped CD8(+) T cells expressed elevated levels of inhibitory receptors and exhibited transcriptomic exhaustion and anergy profiles by gene set enrichment analysis. Dysfunctional, impaired effector differentiation also occurred following immunization of CD4(+) T cell-deficient mice with a poxvirus vector. This study demonstrates that following priming with viral vectors, CD4(+) T cell help is required to promote both the expansion and acquisition of effector functions by CD8(+) T cells, which is accomplished by preventing immediate dysfunction., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Zika virus (ZIKV) is a flavivirus that is responsible for the current epidemic in Brazil and the Americas. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans and mice. The rapid development of a safe and effective ZIKV vaccine is a global health priority, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization with a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a strain of ZIKV involved in the outbreak in northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice. We produced DNA vaccines expressing ZIKV pre-membrane and envelope (prM-Env), as well as a series of deletion mutants. The prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV, as measured by absence of detectable viraemia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and depletion of CD4 and CD8 T lymphocytes in vaccinated mice did not abrogate this protection. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans is likely to be achievable.

Unlabelled: We have recently demonstrated that CD4(+)T cell help is required at the time of adenovirus (Ad) vector immunization for the development of functional CD8(+)T cell responses, but the temporal requirement for CD4(+)T cell help for the induction of antibody responses remains unclear. Here we demonstrate that induction of antibody responses in C57BL/6 mice can occur at a time displaced from the time of Ad vector immunization by depletion of CD4(+)T cells. Transient depletion of CD4(+)T cells at the time of immunization delays the development of antigen-specific antibody responses but does not permanently impair their development or induce tolerance against the transgene. Upon CD4(+)T cell recovery, transgene-specific serum IgG antibody titers develop and reach a concentration equivalent to that in undepleted control animals. These delayed antibody responses exhibit no functional defects with regard to isotype, functional avidity, expansion after boosting immunization, or the capacity to neutralize a simian immunodeficiency virus (SIV) Env-expressing pseudovirus. The development of this delayed transgene-specific antibody response is temporally linked to the expansion of de novo antigen-specific CD4(+)T cell responses, which develop after transient depletion of CD4(+)T cells. These data demonstrate that functional vaccine-elicited antibody responses can be induced even if CD4(+)T cell help is provided at a time markedly separated from the time of vaccination., Importance: CD4(+)T cells have a critical role in providing positive help signals to B cells, which promote robust antibody responses. The paradigm is that helper signals must be provided immediately upon antigen exposure, and their absence results in tolerance against the antigen. Here we demonstrate that, in contrast to the current model that the absence of CD4(+)T cell help at priming results in long-term antibody nonresponsiveness, antibody responses can be induced by adenovirus vector immunization or alum-adjuvanted protein immunization even if CD4(+)T cell help is not provided until >1 month after immunization. These data demonstrate that the time when CD4(+)T cell help signals must be provided is more dynamic and flexible than previously appreciated. These data suggest that augmentation of CD4(+)T cell helper function even after the time of vaccination can enhance vaccine-elicited antibody responses and thereby potentially enhance the immunogenicity of vaccines in immunocompromised individuals., (Copyright © 2016 Provine et al.)

CD4 T cells promote innate and adaptive immune responses, but how vaccine-elicited CD4 T cells contribute to immune protection remains unclear. We evaluated whether induction of virus-specific CD4 T cells by vaccination would protect mice against infection with chronic lymphocytic choriomeningitis virus (LCMV). Immunization with vaccines that selectively induced CD4 T cell responses resulted in catastrophic inflammation and mortality after challenge with a persistent strain of LCMV. Immunopathology required antigen-specific CD4 T cells and was associated with a cytokine storm, generalized inflammation, and multi-organ system failure. Virus-specific CD8 T cells or antibodies abrogated the pathology. These data demonstrate that vaccine-elicited CD4 T cells in the absence of effective antiviral immune responses can trigger lethal immunopathology., (Copyright © 2015, American Association for the Advancement of Science.)

Hexon modification of adenovirus type 5 (Ad5) vectors with the hypervariable regions (HVRs) of Ad48 has been shown to allow Ad5HVR48 vectors to circumvent the majority of the preexisting Ad5-neutralizing antibodies. However, it remains unclear whether modifying hexon HVRs impacts innate or adaptive immune responses elicited by this vector. In this study, we investigated the influence of the HVR substitution of Ad5 on innate and adaptive immune responses following vaccination. Ad5HVR48 displayed an intermediate level of innate immune cytokines and chemokines relative to those of Ad5 and Ad48, consistent with its chimeric nature. Hepatotoxicity was observed after Ad5 immunization but not after Ad5HVR48 or Ad48 immunization. However, the CD8(+) T-cell responses elicited by Ad5HVR48 vectors displayed a partially exhausted phenotype, as evidenced by the sustained expression of programmed death 1 (PD-1), decreased effector-to-central memory conversion, and reduced memory recall responses, similar to those elicited by Ad5 vectors and in contrast to those induced by Ad48 vectors. Taken together, these results indicate that although Ad5HVR48 largely bypasses preexisting Ad5 neutralizing antibodies and shows reduced hepatotoxicity compared to that of Ad5, it induces adaptive immune phenotypes that are functionally exhausted similar to those elicited by Ad5., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Macrophages characterized as M2 and M2-like regulate immune responses associated with immune suppression and healing; however, the relationship of this macrophage subset to CD169+ tissue-resident macrophages and their contribution to shaping alloimmune responses is unknown. Here we identified a population of M2-like tissue-resident macrophages that express high levels of the phosphatidylserine receptor TIM-4 and CD169 (TIM-4hiCD169+). Labeling and tracking of TIM-4hiCD169+ macrophages in mice revealed that this population is a major subset of tissue-resident macrophages, homes to draining LNs following oxidative stress, exhibits an immunoregulatory and hypostimulatory phenotype that is maintained after migration to secondary lymphoid organs, favors preferential induction of antigen-stimulated Tregs, and is highly susceptible to apoptosis. Moreover, CD169+ tissue-resident macrophages were resistant to oxidative stress-induced apoptosis in mice lacking TIM-4. Compared with heart allografts from WT mice, Tim4-/- heart allografts survived much longer and were more easily tolerized by non-immunosuppressed recipients. Furthermore, Tim4-/- allograft survival was associated with the infiltration of Tregs into the graft. Together, our data provide evidence that M2-like TIM-4hiCD169+ tissue-resident macrophages are immunoregulatory and promote engraftment of cardiac allografts, but their influence is diminished by TIM-4-dependent programmed cell death.

Despite the widespread use of replication-incompetent recombinant adenovirus (Ad) vectors as candidate vaccine platforms, the mechanism by which these vectors elicit CD8(+) T cell responses remains poorly understood. Our data demonstrate that induction and maintenance of CD8(+) T cell responses by Ad vector immunization is longitudinally dependent on CD4(+) T cell help for a prolonged period. Depletion of CD4(+) T cells in wild type mice within the first 8 d following Ad immunization resulted in dramatically reduced induction of Ag-specific CD8(+) T cells, decreased T-bet and eomesodermin expression, impaired KLRG1(+) effector differentiation, and atypical expression of the memory markers CD127, CD27, and CD62L. Moreover, these CD8(+) T cells failed to protect against a lethal recombinant Listeria monocytogenes challenge. Depletion of CD4(+) T cells between weeks 1 and 4 following immunization resulted in increased contraction of memory CD8(+) T cells. These data demonstrate a prolonged temporal requirement for CD4(+) T cell help for vaccine-elicited CD8(+) T cell responses in mice. These findings have important implications in the design of vaccines aimed at eliciting CD8(+) T cell responses and may provide insight into the impaired immunogenicity of vaccines in the context of AIDS and other CD4(+) T cell immune deficiencies., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Leptin is an adipose-secreted hormone that plays an important role in both metabolism and immunity. Leptin has been shown to induce Th1-cell polarization and inhibit Th2-cell responses. Additionally, leptin induces Th17-cell responses, inhibits regulatory T (Treg) cells and modulates autoimmune diseases. Here, we investigated whether leptin mediates its activity on T cells by influencing dendritic cells (DCs) to promote Th17 and Treg-cell immune responses in mice. We observed that leptin deficiency (i) reduced the expression of DC maturation markers, (ii) decreased DC production of IL-12, TNF-α, and IL-6, (iii) increased DC production of TGF-β, and (iv) limited the capacity of DCs to induce syngeneic CD4(+) T-cell proliferation. As a consequence of this unique phenotype, DCs generated under leptin-free conditions induced Treg or TH 17 cells more efficiently than DCs generated in the presence of leptin. These data indicate important roles for leptin in DC homeostasis and the initiation and maintenance of inflammatory and regulatory immune responses by DCs., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adipose tissue-derived mesenchymal stem cells (ADSC) exhibit immunosuppressive capabilities both in vitro and in vivo. Their use for therapy in the transplant field is attractive as they could render the use of immunosuppressive drugs unnecessary. The aim of this study was to investigate the effect of ADSC therapy on prolonging skin allograft survival. Animals that were treated with a single injection of donor allogeneic ADSC one day after transplantation showed an increase in donor skin graft survival by approximately one week. This improvement was associated with preserved histological morphology, an expansion of CD4(+) regulatory T cells (Treg) in draining lymph nodes, as well as heightened IL-10 expression and down-regulated IL-17 expression. In vitro, ADSC inhibit naïve CD4(+) T cell proliferation and constrain Th-1 and Th-17 polarization. In summary, infusion of ADSC one day post-transplantation dramatically increases skin allograft survival by inhibiting the Th-17 pathogenic immune response and enhancing the protective Treg immune response. Finally, these data suggest that ADSC therapy will open new opportunities for promoting drug-free allograft survival in clinical transplantation.

Background: Antidepressant-induced sexual dysfunction affects approximately 50% of patients taking antidepressants. Previous research has explored sildenafil's effectiveness in treating various forms of erectile dysfunction, but there is no research supporting sildenafil's use for improving the quality of life for patients with sexual dysfunction linked to antidepressant use. The authors of this article aimed to assess the improvements in quality of life in patients taking sildenafil to treat antidepressant-induced sexual dysfunction., Methods: One hundred and two out of 2,239 male and female patients in the follow-up phase of the Sequenced Treatment Alternatives to Relieve Depression antidepressant trials who complained of sexual dysfunction were given sildenafil, 50 to 100 mg, as needed. After 12 months, we measured patients' change in libido, sexual drive, family relationships, overall well-being, satisfaction with treatment, and overall contentment with items on the 17-item Hamilton Depression Rating Scale, Quality of Life Enjoyment and Satisfaction Questionnaire, 30-item Inventory of Depressive Symptoms, and 12-item Short Form Health Survey., Results: There was a significant association between sildenafil use and improvement in libido and sexual drive by month 6. There was no significant improvement in the quality-of-life scores we examined, but treatment satisfaction and overall contentment increased over time., Conclusions: Despite no direct association with sildenafil use and quality-of-life scores, sildenafil may be a beneficial treatment for antidepressant-induced sexual dysfunction. A double-blind, placebo-controlled study of sildenafil in antidepressant-induced sexual dysfunction is needed to further explore its potential benefits.

Leptin, an adipose-secreted hormone, links metabolism and immunity. Our aim was to determine whether leptin affects the alloimmune response. We used an allogeneic skin transplant model as a means to analyze the allograft immune response in Lep(ob/ob) and wild-type mice. Leptin deficiency results in an increased frequency of Treg and Th2 cells and a prolonged graft survival. These effects of leptin deficiency indicate the importance of leptin and obesity in modulating the allograft immune responses. Our data suggest a possible explanation for the increased susceptibility of hyperleptinemic obese patients to acute and chronic graft rejection., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

To assess whether early changes in Hamilton Depression Rating Scale-17 anxiety/somatization items predict remission in two controlled studies of Hypericum perforatum (St John's wort) versus selective serotonin reuptake inhibitors for major depressive disorder. The Hypericum Depression Trial Study Group (National Institute of Mental Health) randomized 340 patients to Hypericum, sertraline, or placebo for 8 weeks, whereas the Massachusetts General Hospital study randomized 135 patients to Hypericum, fluoxetine, or placebo for 12 weeks. The investigators examined whether remission was associated with early changes in anxiety/somatization symptoms. In the National Institute of Mental Health study, significant associations were observed between remission and early improvement in the anxiety (psychic) item (sertraline arm), somatic (gastrointestinal item; Hypericum arm), and somatic (general) symptoms (placebo arm). None of the three treatment arms of the Massachusetts General Hospital study showed significant associations between anxiety/somatization symptoms and remission. When both study samples were pooled, we found associations for anxiety (psychic; selective serotonin reuptake inhibitors arm), somatic (gastrointestinal), and hypochondriasis (Hypericum arm), and anxiety (psychic) and somatic (general) symptoms (placebo arm). In the entire sample, remission was associated with the improvement in the anxiety (psychic), somatic (gastrointestinal), and somatic (general) items. The number and the type of anxiety/somatization items associated with remission varied depending on the intervention. Early scrutiny of the Hamilton Depression Rating Scale-17 anxiety/somatization items may help to predict remission of major depressive disorder.