Zika purified inactivated virus (ZPIV) vaccine reduced vertical transmission in pregnant immunocompetent mice.

Zika virus (ZIKV) is a significant threat to pregnant women and their fetuses as it can cause severe birth defects and congenital neurodevelopmental disorders, referred to as congenital Zika syndrome (CZS). Thus, a safe and effective ZIKV vaccine for pregnant women to prevent in utero ZIKV infection is of utmost importance. Murine models of ZIKV infection are limited by the fact that immunocompetent mice are resistant to ZIKV infection. As such, interferon-deficient mice have been used in some preclinical studies to test the efficacy of ZIKV vaccine candidates against lethal virus challenge. However, interferon-deficient mouse models have limitations in assessing the immunogenicity of vaccines, necessitating the use of immunocompetent mouse pregnancy models. Using the human stat2 knock-in (hSTAT2KI) mouse pregnancy model, we show that vaccination with a purified formalin-inactivated Zika virus (ZPIV) vaccine prior to pregnancy successfully prevented vertical transmission. In addition, maternal immunity protected offspring against postnatal challenge for up to 28 days. Furthermore, passive transfer of human IgG purified from hyper-immune sera of ZPIV vaccinees prevented maternal and fetal ZIKV infection, providing strong evidence that the neutralizing antibody response may serve as a meaningful correlate of protection.

Protective efficacy of a Zika purified inactivated virus vaccine candidate during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

Impact of prior dengue virus infection on Zika virus infection during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.

Antioxidative and Anti-Inflammatory Activities of Rosebud Extracts of Newly Crossbred Roses.

Oxidative stress and inflammation are basic pathogenic factors involved in tissue injury and pain, as well as acute and chronic diseases. Since long-term uses of synthetic steroids and non-steroidal anti-inflammatory drugs (NSAIDs) cause severe adverse effects, novel effective materials with minimal side effects are required. In this study, polyphenol content and antioxidative activity of rosebud extracts from 24 newly crossbred Korean roses were analyzed. Among them, Pretty Velvet rosebud extract (PVRE) was found to contain high polyphenols and to show in vitro antioxidative and anti-inflammatory activities. In RAW 264.7 cells stimulated with lipopolysaccharide (LPS), PVRE down-regulated mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and thereby decreased nitric oxide (NO) and prostaglandin E2 (PGE2) production. In a subcutaneous air-pouch inflammation model, treatment with PVRE decreased λ-carrageenan-induced tissue exudation, infiltration of inflammatory cells, and inflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß concentrations, as achieved with dexamethasone (a representative steroid). Notably, PVRE also inhibited PGE2, similar to dexamethasone and indomethacin (a representative NSAID). The anti-inflammatory effects of PVRE were confirmed by microscopic findings, attenuating tissue erythema, edema, and inflammatory cell infiltration. These results indicate that PVRE exhibits dual (steroid- and NSAID-like) anti-inflammatory activities by blocking both the iNOS-NO and COX-2-PG pathways, and that PVRE could be a potential candidate as an anti-inflammatory material for diverse tissue injuries.

Efficacy of an inactivated Zika vaccine against virus infection during pregnancy in mice and marmosets.

Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV's protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.

Pre-Clinical Pregnancy Models for Evaluating Zika Vaccines.

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.

Zika virus infection in immunocompetent pregnant mice causes fetal damage and placental pathology in the absence of fetal infection.

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.

Challenges of Vaccine Development for Zika Virus.

The emergence of outbreaks of Zika virus (ZIKV) in Brazil in 2015 was associated with devastating effects on fetal development and prompted a world health emergency and multiple efforts to generate an effective vaccine against infection. There are now more than 40 vaccine candidates in preclinical development and six in clinical trials. Despite similarities with other flaviviruses to which successful vaccines have been developed, such as yellow fever virus and Japanese Encephalitis virus, there are unique challenges to the development and clinical trials of a vaccine for ZIKV.

Anti-nicotine vaccines: Comparison of adjuvanted CRM197 and Qb-VLP conjugate formulations for immunogenicity and function in non-human primates.

Anti-nicotine vaccines comprise nicotine-like haptens conjugated to a carrier protein plus adjuvant(s). Unfortunately, those tested clinically have failed to improve overall long term quit rates. We had shown in mice that carrier, hapten, linker, hapten load (number of haptens per carrier molecule), aggregation and adducts, as well as adjuvants influence the function of antibodies (Ab) induced. Herein, we tested an optimized antigen, NIC7-CRM, comprised of 5-aminoethoxy-nicotine (NIC7) conjugated to genetically detoxified diphtheria toxin (CRM197), with hapten load of ~16, no aggregation (~100% monomer) and minimal adducts. NIC7-CRM was tested in non-human primates (NHP) and compared to NIC-VLP, which has the same hapten and carrier as the clinical-stage CYT002-NicQb but a slightly different linker and lower hapten load. With alum as sole adjuvant, NIC7-CRM was superior to NIC-VLP for Ab titer, avidity and ex vivo function (83% and 27% nicotine binding at 40ng/mL respectively), but equivalent for in vivo function after intravenous [IV] nicotine challenge (brain levels reduced ~10%). CpG adjuvant added to NIC7-CRM/alum further enhanced the Ab responses and both ex vivo function (100% bound) and in vivo function (~80% reduction in brain). Thus, both optimal antigen design and CpG adjuvant were required to achieve a highly functional vaccine. The compelling NHP data with NIC7-CRM with alum/CpG supported human testing, currently underway.

Enhancing immunogenicity of a 3'aminomethylnicotine-DT-conjugate anti-nicotine vaccine with CpG adjuvant in mice and non-human primates.

Tobacco smoking is one of the most preventable causes of morbidity and mortality, but current smoking cessation treatments have relatively poor long term efficacy. Anti-nicotine vaccines offer a novel mechanism of action whereby anti-nicotine antibodies (Ab) in circulation prevent nicotine from entering the brain, thus avoiding the reward mechanisms that underpin nicotine addiction. Since antibody responses are typically long lasting, such vaccines could potentially lead to better long-term smoking cessation outcomes. Clinical trials of anti-nicotine vaccines to date have not succeeded, although there was evidence that very high anti-nicotine Ab titers could lead to improved smoking cessation outcomes, suggesting that achieving higher titers in more subjects might result in better efficacy overall. In this study, we evaluated CpG (TLR9 agonist) and aluminum hydroxide (Al(OH)3) adjuvants with a model anti-nicotine antigen comprising trans-3'aminomethylnicotine (3'AmNic) conjugated to diphtheria toxoid (DT). Anti-nicotine Ab titers were significantly higher in both mice and non-human primates (NHP) when 3'AmNic-DT was administered with CpG/Al(OH)3 than with Al(OH)3 alone, and affinity was enhanced in mice. CpG also improved functional responses, as measured by nicotine brain levels in mice after intravenous administration of radiolabeled nicotine (30% versus 3% without CpG), or by nicotine binding capacity of NHP antisera (15-fold higher with CpG). Further improvement should focus on maximizing Ab function, which takes into account both titer and avidity, and this may require improved conjugate design in addition to adjuvants.

Vaccination with cancer- and HIV infection-associated endogenous retrotransposable elements is safe and immunogenic.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

Differential impact of ageing on cellular and humoral immunity to a persistent murine gamma-herpesvirus.

BACKGROUND: Oncogenic gamma-herpesviruses establish life-long infections in their hosts and control of these latent infections is dependent on continual immune surveillance. Immune function declines with age, raising the possibility that immune control of gamma-herpesvirus infection becomes compromised with increasing age, allowing viral reactivation and/or increased latent load, both of which are associated with the development of malignancies. RESULTS: In this study, we use the experimental mouse gamma-herpesvirus model, gammaHV68, to investigate viral immunity in aged mice. We found no evidence of viral recrudescence or increased latent load in aged latently-infected mice, suggesting that effective immune control of gamma-herpesvirus infection remains intact with ageing. As both cellular and humoral immunity have been implicated in host control of gammaHV68 latency, we independently examined the impact of ageing on gammaHV68-specific CD8 T cell function and antibody responses. Virus-specific CD8 T cell numbers and cytolytic function were not profoundly diminished with age. In contrast, whereas ELISA titers of virus-specific IgG were maintained over time, there was a progressive decline in neutralizing activity. In addition, although aged mice were able to control de novo acute infection with only slightly delayed viral clearance, serum titers of neutralizing antibody were reduced in aged mice as compared to young mice. CONCLUSION: Although there is no obvious loss of immune control of latent virus, these data indicate that ageing has differential impacts on anti-viral cellular and humoral immune protection during persistent gammaHV68 infection. This observation has potential relevance for understanding gamma-herpesvirus immune control during disease-associated or therapeutic immunosuppression.

Perturbation of B cell activation in SLAM-associated protein-deficient mice is associated with changes in gammaherpesvirus latency reservoirs.

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)) interactions with SLAM family proteins play important roles in immune function. SAP-deficient mice have defective B cell function, including impairment of germinal center formation, production of class-switched Ig, and development of memory B cells. B cells are the major reservoir of latency for both EBV and the homologous murine gammaherpesvirus, gammaherpesvirus 68. There is a strong association between the B cell life cycle and viral latency in that the virus preferentially establishes latency in activated germinal center B cells, which provides access to memory B cells, a major reservoir of long-term latency. In the current studies, we have analyzed the establishment and maintenance of gammaHV68 latency in wild-type and SAP-deficient mice. The results show that, despite SAP-associated defects in germinal center and memory B cell formation, latency was established and maintained in memory B cells at comparable frequencies to wild-type mice, although the paucity of memory B cells translated into a 10-fold reduction in latent load. Furthermore, there were defects in normal latency reservoirs within the germinal center cells and IgD(+)"naive" B cells in SAP-deficient mice, showing a profound effect of the SAP mutation on latency reservoirs.

Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo.

Lymphocyte activation gene-3 (LAG-3) is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity. In this study, we used four experimental systems to reevaluate previous suggestions that LAG-3(-/-) mice had no T cell defect. First, LAG-3(-/-) T cells exhibited a delay in cell cycle arrest following in vivo stimulation with the superantigen staphylococcal enterotoxin B resulting in increased T cell expansion and splenomegaly. Second, increased T cell expansion was also observed in adoptive recipients of LAG-3(-/-) OT-II TCR transgenic T cells following in vivo Ag stimulation. Third, infection of LAG-3(-/-) mice with Sendai virus resulted in increased numbers of memory CD4(+) and CD8(+) T cells. Fourth, CD4(+) T cells exhibited a delayed expansion in LAG-3(-/-) mice infected with murine gammaherpesvirus. In summary, these data suggest that LAG-3 negatively regulates T cell expansion and controls the size of the memory T cell pool.

Intact type 1 immunity and immune-associated coagulative responses in mice lacking IFN gamma-inducible fibrinogen-like protein 2.

Fibrinogen-like protein 2 (Fgl2, fibroleukin) is a leukocyte product that exhibits significant homology to secreted proteins of diverse function, including growth factors, lectins, and components of extracellular matrix. Prior studies found that Fgl2 is IFN gamma-inducible, possesses direct coagulant activity, and inhibits T cell proliferation and dendritic cell maturation in vitro. Here, we demonstrate that Fgl2 expression is up-regulated during type 1 immunity in vivo and establish that such up-regulation is IFN gamma-, signal transducer and activation of transcription protein 1-, and IFN response factor 1-dependent. To investigate functional roles for Fgl2 during type 1 immunity, we generated Fgl2-deficient mice. Those animals are born at predicted Mendelian frequencies, appear overtly healthy, and contain normal numbers and frequencies of lymphoid cells. Although Fgl2 is IFN gamma-inducible and putatively regulates T cell activation/proliferation, we demonstrate that Fgl2-deficient and control mice exhibit similar degrees of T cell expansion, immunopathology, and/or pathogen burdens during protozoan (Toxoplasma gondii), bacterial (Yersinia enterocolitica, Listeria monocytogenes, and Mycobacterium tuberculosis), and viral (murine gamma-herpesvirus-68 and Sendai) infections. Fgl2-deficient mice also reject allografts with similar kinetics as control mice. Moreover, despite prior reports that Fgl2 functions as a procoagulant enzyme, we demonstrate that Fgl2-deficient and control mice produce similar levels of fibrin, a product of the coagulation cascade, during T. gondii infection and allograft rejection. Together, our findings suggest that Fgl2, although highly conserved and IFN gamma-inducible, is not a critical mediator of either type 1 immunity or immune-associated coagulant activity.

T cell reactivity during infectious mononucleosis and persistent gammaherpesvirus infection in mice.

Intranasal infection of mice with murine gammaherpesvirus 68 causes a dramatic increase in numbers of activated CD8(+) T cells in the blood, analogous in many respects to EBV-induced infectious mononucleosis in humans. In the mouse model, this lymphocytosis has two distinct components: an early, conventional virus-specific CD8(+) T cell response, and a later response characterized by a dramatic increase among CD8(+) T cells that bear Vbeta4(+) TCRs. We previously demonstrated that Vbeta4(+)CD8(+) T cells recognize an uncharacterized ligand expressed on latently infected B cells in an MHC-independent manner. The frequency of Vbeta4(+)CD8(+) T cells increases dramatically following the peak of viral latency in the spleen. In the current studies, we show that elevated Vbeta4(+)CD8(+) T cell levels are sustained long-term in persistently infected mice, apparently a consequence of continued ligand expression. In addition, we show that Vbeta4(+)CD8(+) T cells can acquire effector functions, including cytotoxicity and the capacity to secrete IFN-gamma, although they have an atypical activation profile compared with well-characterized CD8(+) T cells specific for conventional viral epitopes. The characteristics of Vbeta4(+)CD8(+) T cells (potential effector function, stimulation by latently infected B cells, and kinetics of expansion) suggested that this dominant T cell response plays a key role in the immune control of latent virus. However, Ab depletion and adoptive transfer studies show that Vbeta4(+)CD8(+) T cells are not essential for this function. This murine model of infection may provide insight into the role of unusual populations of activated T cells associated with persistent viral infections.

A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro.

Natural rubber (cis-1,4-polyisoprene) is an isoprenoid compound produced exclusively in plants by the action of rubber transferase. Despite a keen interest in revealing the mechanisms of rubber chain elongation and chain length determination, the molecular nature of rubber transferase has not yet been identified. A recent report has revealed that a 24 kDa protein tightly associated with the small rubber particles of Hevea brasiliensis, therefore designated small rubber particle protein (SRPP), plays a positive role in rubber biosynthesis. Since guayule (Parthenium argentatum Gray) produces natural rubber similar in size to H. brasiliensis, it is of critical interest to investigate whether guayule contains a similar protein to the SRPP. A cDNA clone has been isolated in guayule that shares a sequence homology with the SRPP, thus designated guayule homologue of SRPP (GHS), and the catalytic function of the protein was characterized. Sequence analysis revealed that the GHS is highly homologous in several conserved regions to the SRPP (50% identity). In vitro functional analysis of the recombinant protein overexpressed in E. coli revealed that the GHS plays a positive role in isopentenyl diphosphate incorporation into high molecular weight rubbers as SRPP does. These results indicate that guayule and Hevea rubber trees contain a protein that is similar in its amino acid sequence and plays a role in isopentenyl diphosphate incorporation in vitro, implying that it contributes to the enhancement of rubber biosynthetic activity in rubber trees.

Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis.

This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.

Maintenance of long term gamma-herpesvirus B cell latency is dependent on CD40-mediated development of memory B cells.

It has been proposed that the gamma-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40(+) and CD40(-) B cells from CD40(+)CD40(-) mixed bone marrow chimera mice after infection with a murine gamma-herpesvirus, MHV-68. CD40(+) B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40(-) B cells and preferentially maintained in the long-lived, isotype-switched CD40(+) B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.