The role of vaccination route with an adenovirus-vectored vaccine in protection, viral control, and transmission in the SARS-CoV-2/K18-hACE2 mouse infection model.

Introduction: Vaccination is the most effective mechanism to prevent severe COVID-19. However, breakthrough infections and subsequent transmission of SARS-CoV-2 remain a significant problem. Intranasal vaccination has the potential to be more effective in preventing disease and limiting transmission between individuals as it induces potent responses at mucosal sites. Methods: Utilizing a replication-deficient adenovirus serotype 5-vectored vaccine expressing the SARS-CoV-2 RBD (AdCOVID) in homozygous and heterozygous transgenic K18-hACE2, we investigated the impact of the route of administration on vaccine immunogenicity, SARS-CoV-2 transmission, and survival. Results: Mice vaccinated with AdCOVID via the intramuscular or intranasal route and subsequently challenged with SARS-CoV-2 showed that animals vaccinated intranasally had improved cellular and mucosal antibody responses. Additionally, intranasally vaccinated animals had significantly better viremic control, and protection from lethal infection compared to intramuscularly vaccinated animals. Notably, in a novel transmission model, intranasal vaccination reduced viral transmission to naïve co-housed mice compared to intramuscular vaccination. Discussion: Our data provide convincing evidence for the use of intranasal vaccination in protecting against SARS-CoV-2 infection and transmission.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

The Ability of Zika virus Intravenous Immunoglobulin to Protect From or Enhance Zika Virus Disease.

The closely related flaviviruses, dengue and Zika, cause significant human disease throughout the world. While cross-reactive antibodies have been demonstrated to have the capacity to potentiate disease or mediate protection during flavivirus infection, the mechanisms responsible for this dichotomy are still poorly understood. To understand how the human polyclonal antibody response can protect against, and potentiate the disease in the context of dengue and Zika virus infection we used intravenous hyperimmunoglobulin (IVIG) preparations in a mouse model of the disease. Three IVIGs (ZIKV-IG, Control-Ig and Gamunex®) were evaluated for their ability to neutralize and/or enhance Zika, dengue 2 and 3 viruses in vitro. The balance between virus neutralization and enhancement provided by the in vitro neutralization data was used to predict the IVIG concentrations which could protect or enhance Zika, and dengue 2 disease in vivo. Using this approach, we were able to define the unique in vivo dynamics of complex polyclonal antibodies, allowing for both enhancement and protection from flavivirus infection. Our results provide a novel understanding of how polyclonal antibodies interact with viruses with implications for the use of polyclonal antibody therapeutics and the development and evaluation of the next generation flavivirus vaccines.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Selective estrogen receptor modulator, tamoxifen, inhibits Zika virus infection.

Zika virus (ZIKV) is an arbovirus belonging to the flaviviridae family with a risk assessment that has been increasing in recent years and was labeled a global health emergency by the World Health Organization in 2016. There are currently no Food and Drug Administration-approved treatment options available for ZIKV, so expeditious development of treatment options is urgent. To expedite this process, an on-market drug, tamoxifen (TAM), was selected as a promising candidate for repurposing due to its wide range of biological activities and because it has already been shown to possess activity against hepatitis C virus, a flavivirus in a separate genus. Anti-ZIKV activity of TAM was assessed by compound screens using an infectious virus and mechanistic details were gleaned from time of addition and virucidal studies. TAM and an active metabolite, 4-hydroxytamoxifen (TAM-OH), both showed promising antiviral activity (EC50 ≈9 and 5 µM, respectively) in initial compound screening and up to 8-h postinfection, though the virucidal assay indicated that they do not possess any direct virucidal activity. Additionally, TAM was assessed for its activity against ZIKV in the human male germ cell line, SEM-1, due to the sexually transmitted nature of ZIKV owing to its extended survival times in germ cells. Virus titers show diminished replication of ZIKV over 7 days compared to controls. These data indicate that TAM has the potential to be repurposed as an anti-ZIKV therapeutic and warrants further investigation.

Pre-existing T Cell Memory against Zika Virus.

The mosquito-borne Zika virus (ZIKV) has spread rapidly into regions where dengue virus (DENV) is endemic, and flavivirus cross-reactive T cell responses have been observed repeatedly in animal models and in humans. Preexisting cellular immunity to DENV is thought to contribute to protection in subsequent ZIKV infection, but the epitope targets of cross-reactive T cell responses have not been comprehensively identified. Using human blood samples from the regions of Nicaragua and Sri Lanka where DENV is endemic that were collected before the global spread of ZIKV in 2016, we employed an in vitro expansion strategy to map ZIKV T cell epitopes in ZIKV-unexposed, DENV-seropositive donors. We identified 93 epitopes across the ZIKV proteome, and we observed patterns of immunodominance that were dependent on antigen size and sequence identity to DENV. We confirmed the immunogenicity of these epitopes through a computational HLA binding analysis, and we showed that cross-reactive T cells specifically recognize ZIKV peptides homologous to DENV sequences. We also found that these CD4 responses were derived from the memory T cell compartment. These data have implications for understanding the dynamics of flavivirus-specific T cell immunity in areas of endemicity.IMPORTANCE Multiple flaviviruses, including Zika virus (ZIKV) and the four serotypes of dengue virus (DENV), are prevalent in the same large tropical and equatorial areas, which are inhabited by hundreds of millions of people. The interplay of DENV and ZIKV infection is especially relevant, as these two viruses are endemic in largely overlapping regions, have significant sequence similarity, and share the same arthropod vector. Here, we define the targets of preexisting immunity to ZIKV in unexposed subjects in areas where dengue is endemic. We demonstrate that preexisting immunity to DENV could shape ZIKV-specific responses, and DENV-ZIKV cross-reactive T cell populations can be expanded by stimulation with ZIKV peptides. The issue of potential ZIKV and DENV cross-reactivity is of relevance for understanding patterns of natural immunity, as well as for the development of diagnostic tests and vaccines.

A Dengue Virus Serotype 1 mRNA-LNP Vaccine Elicits Protective Immune Responses.

Dengue virus (DENV) is the most common vector-borne viral disease, with nearly 400 million worldwide infections each year concentrated in the tropical and subtropical regions of the world. Severe dengue complications are often associated with a secondary heterotypic infection of one of the four circulating serotypes. In this scenario, humoral immune responses targeting cross-reactive, poorly neutralizing epitopes can lead to increased infectivity of susceptible cells via antibody-dependent enhancement (ADE). In this way, antibodies produced in response to infection or vaccination are capable of contributing to enhanced disease in subsequent infections. Currently, there are no available therapeutics to combat DENV disease, and there is an urgent need for a safe and efficacious vaccine. Here, we developed a nucleotide-modified mRNA vaccine encoding the membrane and envelope structural proteins from DENV serotype 1 encapsulated in lipid nanoparticles (prM/E mRNA-LNP). Vaccination of mice elicited robust antiviral immune responses comparable to viral infection, with high levels of neutralizing antibody titers and antiviral CD4+ and CD8+ T cells. Immunocompromised AG129 mice vaccinated with the prM/E mRNA-LNP vaccine were protected from a lethal DENV challenge. Vaccination with either a wild-type vaccine or a vaccine with mutations in the immunodominant fusion loop epitope elicited equivalent humoral and cell-mediated immune responses. Neutralizing antibodies elicited by the vaccine were sufficient to protect against a lethal challenge. Both vaccine constructs demonstrated serotype-specific immunity with minimal serum cross-reactivity and reduced ADE in comparison to a live DENV1 viral infection.IMPORTANCE With 400 million worldwide infections each year, dengue is the most common vector-borne viral disease. Forty percent of the world's population is at risk, with dengue experiencing consistent geographic spread over the years. With no therapeutics available and vaccines performing suboptimally, the need for an effective dengue vaccine is urgent. Here, we develop and characterize a novel mRNA vaccine encoding the dengue serotype 1 envelope and premembrane structural proteins that is delivered via a lipid nanoparticle. Our DENV1 prM/E mRNA-LNP vaccine induces neutralizing antibody and cellular immune responses in immunocompetent mice and protects an immunocompromised mouse from a lethal DENV challenge. Existing antibodies against dengue can enhance subsequent infections via antibody-dependent enhancement (ADE). Importantly our vaccine induced only serotype-specific immune responses and did not induce ADE.

Heterotypic immunity against vaccinia virus in an HLA-B*07:02 transgenic mousepox infection model.

Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8+ T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8+ T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8+ T cells were not recognized by VACV-reactive CD8+ T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R70-78 variant of the dominant VACV B8R70-78 epitope. C4R70-78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

Generation and characterization of an Il2rg knockout Syrian hamster model for XSCID and HAdV-C6 infection in immunocompromised patients.

Model animals are indispensable for the study of human diseases, and in general, of complex biological processes. The Syrian hamster is an important model animal for infectious diseases, behavioral science and metabolic science, for which more experimental tools are becoming available. Here, we describe the generation and characterization of an interleukin-2 receptor subunit gamma (Il2rg) knockout (KO) Syrian hamster strain. In humans, mutations in IL2RG can result in a total failure of T and natural killer (NK) lymphocyte development and nonfunctional B lymphocytes (X-linked severe combined immunodeficiency; XSCID). Therefore, we sought to develop a non-murine model to study XSCID and the infectious diseases associated with IL2RG deficiency. We demonstrated that the Il2rg KO hamsters have a lymphoid compartment that is greatly reduced in size and diversity, and is impaired in function. As a result of the defective adaptive immune response, Il2rg KO hamsters developed a more severe human adenovirus infection and cleared virus less efficiently than immune competent wild-type hamsters. Because of this enhanced virus replication, Il2rg KO hamsters developed more severe adenovirus-induced liver pathology than wild-type hamsters. This novel hamster strain will provide researchers with a new tool to investigate human XSCID and its related infections.

Immunogenicity and Efficacy of a Recombinant Human Adenovirus Type 5 Vaccine against Zika Virus.

Zika virus (ZIKV) is a significant public health concern due to the pathogen's ability to be transmitted by either mosquito bite or sexual transmission, allowing spread to occur throughout the world. The potential consequences of ZIKV infection to human health, specifically neonates, necessitates the development of a safe and effective Zika virus vaccine. Here, we developed an intranasal Zika vaccine based upon the replication-deficient human adenovirus serotype 5 (hAd5) expressing ZIKV pre-membrane and envelope protein (hAd5-ZKV). The hAd5-ZKV vaccine is able to induce both cell-mediated and humoral immune responses to ZIKV epitopes. Importantly, this vaccine generated CD8+ T cells specific for a dominant ZIKV T cell epitope and is shown to be protective against a ZIKV challenge by using a pre-clinical model of ZIKV disease. We also demonstrate that the vaccine expresses pre-membrane and envelope protein in a confirmation recognized by ZIKV experienced individuals. Our studies demonstrate that this adenovirus-based vaccine expressing ZIKV proteins is immunogenic and protective in mice, and it encodes ZIKV proteins in a conformation recognized by the human antibody repertoire.

The small molecule AZD6244 inhibits dengue virus replication in vitro and protects against lethal challenge in a mouse model.

Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 105 PFU of D2S20. The effectiveness of AZD6244 was observed even when the treatment of infected animals was initiated 1-2 days postinfection. This was also followed by a reduction in viral copy number in both the serum and the spleen. There was also an increase in IL-1ß and TNF-α levels in mice that were infected with D2S20 and treated with AZD6244 in comparison to infected mice that were treated with the vehicle only. These data demonstrate the potential of AZD6244 as a new therapeutic agent to treat DENV infection and possibly other flavivirus diseases.

CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.

Zika virus (ZIKV) has gained worldwide attention since it emerged, and a global effort is underway to understand the correlates of protection and develop diagnostics to identify rates of infection. As new therapeutics and vaccine approaches are evaluated in clinical trials, additional effort is focused on identifying the adaptive immune correlates of protection against ZIKV disease. To aid in this endeavor we have begun to dissect the role of CD4+T cells in the protection against neuroinvasive ZIKV disease. We have identified an important role for CD4+T cells in protection, demonstrating that in the absence of CD4+T cells mice have more severe neurological sequela and significant increases in viral titers in the central nervous system (CNS). The transfer of CD4+T cells from ZIKV immune mice protect type I interferon receptor deficient animals from a lethal challenge; showing that the CD4+T cell response is necessary and sufficient for control of ZIKV disease. Using a peptide library spanning the complete ZIKV polyprotein, we identified both ZIKV-encoded CD4+T cell epitopes that initiate immune responses, and ZIKV specific CD4+T cell receptors that recognize these epitopes. Within the ZIKV antigen-specific TCRß repertoire, we uncovered a high degree of diversity both in response to a single epitope and among different mice responding to a CD4+T cell epitope. Overall this study identifies a novel role for polyfunctional and polyclonal CD4+T cells in providing protection against ZIKV infection and highlights the need for vaccines to develop robust CD4+T cell responses to prevent ZIKV neuroinvasion and limit replication within the CNS.

Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus.

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis.

Isolation and Characterization of Broad and Ultrapotent Human Monoclonal Antibodies with Therapeutic Activity against Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/ml), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development.

Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection.

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity.

Development of a highly protective combination monoclonal antibody therapy against Chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar(-/-) ) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.

The interferon-inducible gene viperin restricts West Nile virus pathogenesis.

Type I interferon (IFN) signaling coordinates an early antiviral program in infected and uninfected cells by inducing IFN-stimulated genes (ISGs) that modulate viral entry, replication, and assembly. However, the specific antiviral functions in vivo of most ISGs remain unknown. Here, we examined the contribution of the ISG viperin to the control of West Nile virus (WNV) in genetically deficient cells and mice. While modest increases in levels of WNV replication were observed for primary viperin(-/-) macrophages and dendritic cells, no appreciable differences were detected in deficient embryonic cortical neurons or fibroblasts. In comparison, viperin(-/-) adult mice infected with WNV via the subcutaneous or intracranial route showed increased lethality and/or enhanced viral replication in central nervous system (CNS) tissues. In the CNS, viperin expression was induced in both WNV-infected and adjacent uninfected cells, including activated leukocytes at the site of infection. Our experiments suggest that viperin restricts the infection of WNV in a tissue- and cell-type-specific manner and may be an important ISG for controlling viral infections that cause CNS disease.

Interferon regulatory factor-1 (IRF-1) shapes both innate and CD8(+) T cell immune responses against West Nile virus infection.

Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-αß expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1(-/-) mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1(-/-) cells and mice. IRF-1(-/-) mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1(-/-) macrophages supported enhanced WNV replication but infection was unaltered in IRF-1(-/-) fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8(+) T cell expansion. Although markedly fewer CD8(+) T cells were observed in naïve animals as described previously, remarkably, IRF-1(-/-) mice rapidly expanded their pool of WNV-specific cytolytic CD8(+) T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8(+) T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8(+) T cell response.

Repeated in vivo stimulation of T and B cell responses in old mice generates protective immunity against lethal West Nile virus encephalitis.

Older adults exhibit higher morbidity and mortality from infectious diseases compared with those of the general population. The introduction and rapid spread of West Nile virus (WNV) throughout the continental United States since 1999 has highlighted the challenge of protecting older adults against emerging pathogens: to this day there is no therapy or vaccine approved for human use against West Nile encephalitis. In this study, we describe the characterization of T and B cell responses in old mice after vaccination with RepliVAX WN, a novel West Nile encephalitis vaccine based on single-cycle flavivirus particles. In adult mice, RepliVAX WN induced robust and long-lasting CD4(+) and CD8(+) T cell and Ab (B cell) responses against natural WNV epitopes, similar to those elicited by primary WNV infection. Primary and memory T and B cell responses in old mice against RepliVAX WN vaccination were significantly lower than those seen in younger mice, similar to the response of old mice to infection with WNV. Surprisingly, both the quality and the quantity of the recall Ab and T cell responses in vaccinated old mice were improved to equal or exceed those in adult animals. Moreover, these responses together (but not individually) were sufficient to protect both old and adult mice from severe WNV disease upon challenge. Therefore, at least two cycles of in vivo restimulation are needed for selection and expansion of protective lymphocytes in older populations, and live, single-cycle virus vaccines that stimulate both cellular and humoral immunity can protect older individuals against severe viral disease.