Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding are not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in enzyme-linked immunosorbent assays (ELISAs) and depended on heparan sulfate for efficient cell binding and infection, we observed some variation by strain. Enzymatic removal of cell surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection.IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.

Transgenic Wheat Harboring an RNAi Element Confers Dual Resistance Against Synergistically Interacting Wheat Streak Mosaic Virus and Triticum Mosaic Virus.

Wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV) are economically important viruses of wheat (Triticum aestivum L.), causing significant yield losses in the Great Plains region of the United States. These two viruses are transmitted by wheat curl mites, which often leads to mixed infections with synergistic interaction in grower fields that exacerbates yield losses. Development of dual-resistant wheat lines would provide effective control of these two viruses. In this study, a genetic resistance strategy employing an RNA interference (RNAi) approach was implemented by assembling a hairpin element composed of a 202-bp (404-bp in total) stem sequence of the NIb (replicase) gene from each of WSMV and TriMV in tandem and of an intron sequence in the loop. The derived RNAi element was cloned into a binary vector and was used to transform spring wheat genotype CB037. Phenotyping of T1 lineages across eight independent transgenic events for resistance revealed that i) two of the transgenic events provided resistance to WSMV and TriMV, ii) four events provided resistance to either WSMV or TriMV, and iii) no resistance was found in two other events. T2 populations derived from the two events classified as dual-resistant were subsequently monitored for stability of the resistance phenotype through the T4 generation. The resistance phenotype in these events was temperature-dependent, with a complete dual resistance at temperatures ≥25°C and an increasingly susceptible response at temperatures below 25°C. Northern blot hybridization of total RNA from transgenic wheat revealed that virus-specific small RNAs (vsRNAs) accumulated progressively with an increase in temperature, with no detectable levels of vsRNA accumulation at 20°C. Thus, the resistance phenotype of wheat harboring an RNAi element was correlated with accumulation of vsRNAs, and the generation of vsRNAs can be used as a molecular marker for the prediction of resistant phenotypes of transgenic plants at a specific temperature.

Asymmetry in Synergistic Interaction Between Wheat streak mosaic virus and Triticum mosaic virus in Wheat.

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV), distinct members in the family Potyviridae, are economically important wheat-infecting viruses in the Great Plains region. Previously, we reported that coinfection of wheat by WSMV and TriMV caused disease synergism with increased concentration of both viruses. The mechanisms of synergistic interaction between WSMV and TriMV and the effects of prior infection of wheat by either of these "synergistically interacting partner" (SIP) viruses on the establishment of local and systemic infection by the other SIP virus are not known. In this study, using fluorescent protein-tagged viruses, we found that prior infection of wheat by WSMV or TriMV negatively affected the onset and size of local foci elicited by subsequent SIP virus infection compared with those in buffer-inoculated wheat. These data revealed that prior infection of wheat by an SIP virus has no measurable advantage for another SIP virus on the initiation of infection and cell-to-cell movement. In TriMV-infected wheat, WSMV exhibited accelerated long-distance movement and increased accumulation of genomic RNAs compared with those in buffer-inoculated wheat, indicating that TriMV-encoded proteins complemented WSMV for efficient systemic infection. In contrast, TriMV displayed delayed systemic infection in WSMV-infected wheat, with fewer genomic RNA copies in early stages of infection compared with those in buffer-inoculated wheat. However, during late stages of infection, TriMV accumulation in WSMV-infected wheat increased rapidly with accelerated long-distance movement compared with those in buffer-inoculated wheat. Taken together, these data suggest that interactions between synergistically interacting WSMV and TriMV are asymmetrical; thus, successful establishment of synergistic interaction between unrelated viruses will depend on the order of infection of plants by SIP viruses.

Combination Adenovirus and Protein Vaccines Prevent Infection or Reduce Viral Burden after Heterologous Clade C Simian-Human Immunodeficiency Virus Mucosal Challenge.

HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.

saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern.

Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12 months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains.

CD4+ T Cell Responses to Toxoplasma gondii Are a Double-Edged Sword.

CD4+ T cells have been found to play critical roles in the control of both acute and chronic Toxoplasma infection. Previous studies identified a protective role for the Toxoplasma CD4+ T cell-eliciting peptide AS15 (AVEIHRPVPGTAPPS) in C57BL/6J mice. Herein, we found that immunizing mice with AS15 combined with GLA-SE, a TLR-4 agonist in emulsion adjuvant, can be either helpful in protecting male and female mice at early stages against Type I and Type II Toxoplasma parasites or harmful (lethal with intestinal, hepatic, and spleen pathology associated with a storm of IL6). Introducing the universal CD4+ T cell epitope PADRE abrogates the harmful phenotype of AS15. Our findings demonstrate quantitative and qualitative features of an effective Toxoplasma-specific CD4+ T cell response that should be considered in testing next-generation vaccines against toxoplasmosis. Our results also are cautionary that individual vaccine constituents can cause severe harm depending on the company they keep.

Relative distribution of West Nile virus RNA in blood compartments: implications for blood donor nucleic acid amplification technology screening.

BACKGROUND: Despite implementation of targeted individual-donor nucleic acid test (NAT) screening of blood donors for West Nile virus (WNV), three "breakthrough" WNV transfusion transmission cases were reported (2004-2008), suggesting that current plasma-based assays are unable to detect all WNV-infectious donations. A 2007 report found that 19 of 20 red blood cell components from WNV-infected donors contained 1 log higher viral load than plasma components. This study's aim was to further establish the value of screening whole blood relative to plasma for WNV RNA by generating differential viral loads on paired samples derived from blood screening tubes. STUDY DESIGN AND METHODS: WNV RNA-positive donors identified by routine NAT screening were enrolled and quantitative viral data were generated using cross-sectional (index-donation) and longitudinal (follow-up) specimens. A real-time reverse transcription-polymerase chain reaction viral load assay was used on both study sample sets and replicate qualitative NAT screening assays were also used on the longitudinal study samples. RESULTS: For the cross-sectional study, seronegative index donations (n = 29) had WNV RNA concentrations fourfold higher in plasma than in whole blood, whereas for seropositive donations (n = 13), the WNV RNA concentrations were 10-fold higher in whole blood than in plasma. All 10 longitudinal study participants were seropositive throughout the follow-up study; whole blood viral load was consistently greater than plasma viral load (mean difference, 343 copies; p < 0.001) up to 200 days after index. CONCLUSION: The improved sensitivity of WNV NAT using whole blood instead of plasma was confirmed, but appears to be limited to better detection in seropositive stages. However, the implication of these findings for blood screening requires further study to establish the infectivity of persistent whole blood viremia.

Cellular immune response to cryptic epitopes during therapeutic gene transfer.

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8(+) T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8(+) CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.

Zika virus-like particle vaccine fusion loop mutation increases production yield but fails to protect AG129 mice against Zika virus challenge.

Zika virus (ZIKV) is a mosquito-borne flavivirus with maternal infection associated with preterm birth, congenital malformations, and fetal death, and adult infection associated with Guillain-Barré syndrome. Recent widespread endemic transmission of ZIKV and the potential for future outbreaks necessitate the development of an effective vaccine. We developed a ZIKV vaccine candidate based on virus-like-particles (VLPs) generated following transfection of mammalian HEK293T cells using a plasmid encoding the pre-membrane/membrane (prM/M) and envelope (E) structural protein genes. VLPs were collected from cell culture supernatant and purified by column chromatography with yields of approximately 1-2mg/L. To promote increased particle yields, a single amino acid change of phenylalanine to alanine was made in the E fusion loop at position 108 (F108A) of the lead VLP vaccine candidate. This mutation resulted in a modest 2-fold increase in F108A VLP production with no detectable prM processing by furin to a mature particle, in contrast to the lead candidate (parent). To evaluate immunogenicity and efficacy, AG129 mice were immunized with a dose titration of either the immature F108A or lead VLP (each alum adjuvanted). The resulting VLP-specific binding antibody (Ab) levels were comparable. However, geometric mean neutralizing Ab (nAb) titers using a recombinant ZIKV reporter were significantly lower with F108A immunization compared to lead. After virus challenge, all lead VLP-immunized groups showed a significant 3- to 4-Log10 reduction in mean ZIKV RNAemia levels compared with control mice immunized only with alum, but the RNAemia reduction of 0.5 Log10 for F108A groups was statistically similar to the control. Successful viral control by the lead VLP candidate following challenge supports further vaccine development for this candidate. Notably, nAb titer levels in the lead, but not F108A, VLP-immunized mice inversely correlated with RNAemia. Further evaluation of sera by an in vitro Ab-dependent enhancement assay demonstrated that the F108A VLP-induced immune sera had a significantly higher capacity to promote ZIKV infection in FcγR-expressing cells. These data indicate that a single amino acid change in the fusion loop resulted in increased VLP yields but that the immature F108A particles were significantly diminished in their capacity to induce nAbs and provide protection against ZIKV challenge.

Safety and immunogenicity of PXVX0317, an aluminium hydroxide-adjuvanted chikungunya virus-like particle vaccine: a randomised, double-blind, parallel-group, phase 2 trial.

BACKGROUND: Chikungunya virus (CHIKV) disease is an ongoing public health threat. We aimed to evaluate the safety and immunogenicity of PXVX0317, an aluminium hydroxide-adjuvanted formulation of a CHIKV virus-like particle (VLP) vaccine. METHODS: This randomised, double-blind, parallel-group, phase 2 trial was conducted at three clinical trial centres in the USA. Eligible participants were healthy CHIKV-naïve adults aged 18-45 years. Participants were stratified by site and randomly assigned (1:1:1:1:1:1:1:1) to one of the eight vaccination groups using a block size of 16. Group 1 received two doses of unadjuvanted PXVX0317 28 days apart (2 × 20 µg; standard); all other groups received adjuvanted PXVX0317: groups 2-4 received two doses 28 days apart (2 × 6 µg [group 2], 2 × 10 µg [group 3], or 2 × 20 µg [group 4]; standard); group 4 also received a booster dose 18 months after the first active injection (40 µg; standard plus booster); groups 5-7 received two doses 14 days apart (2 × 6 µg [group 5], 2 × 10 µg [group 6], or 2 × 20 µg [group 7]; accelerated); and group 8 received one dose (1 × 40 µg; single). The primary endpoint was the geometric mean titre of anti-CHIKV neutralising antibody on day 57 (28 days after the last vaccination), assessed in the immunogenicity-evaluable population. Additionally, we assessed safety. This trial is registered at ClinicalTrials.gov, NCT03483961. FINDINGS: This trial was conducted from April 18, 2018, to Sept 21, 2020; 468 participants were assessed for eligibility. Of these, 415 participants were randomly assigned to eight groups (n=53 in groups 1, 5, and 6; n=52 in groups 2 and 8; n=51 in groups 3 and 7; and n=50 in group 4) and 373 were evaluable for immunogenicity. On day 57, serum neutralising antibody geometric mean titres were 2057·0 (95% CI 1584·8-2670·0) in group 1, 1116·2 (852·5-1461·4; p=0·0015 vs group 1 used as a reference) in group 2, 1465·3 (1119·1-1918·4; p=0·076) in group 3, 2023·8 (1550·5-2641·7; p=0·93) in group 4, 920·1 (710·9-1190·9; p<0·0001) in group 5, 1206·9 (932·4-1562·2; p=0·0045) in group 6, 1562·8 (1204·1-2028·3; p=0·14) in group 7, and 1712·5 (1330·0-2205·0; p=0·32) in group 8. In group 4, a booster dose increased serum neutralising antibody geometric mean titres from 215·7 (95% CI 160·9-289·1) on day 547 to 10 941·1 (7378·0-16 225·1) on day 575. Durability of the immune response (evaluated in groups 1, 4, and 8) was shown up to 2 years. The most common solicited adverse event was pain at the injection site, reported in 12 (23%) of 53 participants who received the unadjuvanted vaccine (group 1) and 111 (31%) of 356 who received the adjuvanted vaccine. No vaccine-related serious adverse events were reported. INTERPRETATION: PXVX0317 was well tolerated and induced a robust and durable serum neutralising antibody immune response against CHIKV up to 2 years. A single 40 µg injection of adjuvanted PXVX0317 is being further investigated in phase 3 clinical trials (NCT05072080 and NCT05349617). FUNDING: Emergent BioSolutions.

Differential V2-directed antibody responses in non-human primates infected with SHIVs or immunized with diverse HIV vaccines.

V2p and V2i antibodies (Abs) that are specific for epitopes in the V1V2 region of the HIV gp120 envelope (Env) do not effectively neutralize HIV but mediate Fc-dependent anti-viral activities that have been correlated with protection from, or control of HIV, SIV and SHIV infections. Here, we describe a novel molecular toolbox that allows the discrimination of antigenically and functionally distinct polyclonal V2 Ab responses. We identify different patterns of V2 Ab induction by SHIV infection and three separate vaccine regimens that aid in fine-tuning an optimized immunization protocol for inducing V2p and V2i Abs. We observe no, or weak and sporadic V2p and V2i Abs in non-vaccinated SHIV-infected NHPs, but strong V2p and/or V2i Ab responses after immunization with a V2-targeting vaccine protocol. The V2-focused vaccination is superior to both natural infection and to immunization with whole Env constructs for inducing functional V2p- and V2i-specific responses. Strikingly, levels of V2-directed Abs correlate inversely with Abs specific for peptides of V3 and C5. These data demonstrate that a V1V2-targeting vaccine has advantages over the imprecise targeting of SIV/SHIV infections and of whole Env-based immunization regimens for inducing a more focused functional V2p- and V2i-specific Ab response.

A multivalent vaccination strategy for the prevention of Old World arenavirus infection in humans.

Arenaviruses cause severe human disease ranging from aseptic meningitis following lymphocytic choriomeningitis virus (LCMV) infection to hemorrhagic fever syndromes following infection with Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Machupo virus (MACV), Sabia virus (SABV), or Whitewater Arroyo virus (WWAV). Cellular immunity, chiefly the CD8(+) T-cell response, plays a critical role in providing protective immunity following infection with the Old World arenaviruses LASV and LCMV. In the current study, we evaluated whether HLA class I-restricted epitopes that are cross-reactive among pathogenic arenaviruses could be identified for the purpose of developing an epitope-based vaccination approach that would cross-protect against multiple arenaviruses. We were able to identify a panel of HLA-A*0201-restricted peptides derived from the same region of the glycoprotein precursor (GPC) of LASV (GPC spanning residues 441 to 449 [GPC(441-449)]), LCMV (GPC(447-455)), JUNV (GPC(429-437)), MACV (GPC(444-452)), GTOV (GPC(427-435)), and WWAV (GPC(428-436)) that displayed high-affinity binding to HLA-A*0201 and were recognized by CD8(+) T cells in a cross-reactive manner following LCMV infection or peptide immunization of HLA-A*0201 transgenic mice. Immunization of HLA-A*0201 mice with the Old World peptide LASV GPC(441-449) or LCMV GPC(447-455) induced high-avidity CD8(+) T-cell responses that were able to kill syngeneic target cells pulsed with either LASV GPC(441-449) or LCMV GPC(447-455) in vivo and provided significant protection against viral challenge with LCMV. Through this study, we have demonstrated that HLA class I-restricted, cross-reactive epitopes exist among diverse arenaviruses and that individual epitopes can be utilized as effective vaccine determinants for multiple pathogenic arenaviruses.

A multivalent and cross-protective vaccine strategy against arenaviruses associated with human disease.

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.

Differential Synergistic Interactions Among Four Different Wheat-Infecting Viruses.

Field-grown wheat (Triticum aestivum L.) plants can be co-infected by multiple viruses, including wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV). These viruses belong to four different genera in three different families and are, hence, genetically divergent. However, the impact of potential co-infections with two, three, or all four of them on the viruses themselves, as well as the wheat host, has yet to be examined. This study examined bi-, tri-, and quadripartite interactions among these viruses in wheat for disease development and accumulation of viral genomic RNAs, in comparison with single virus infections. Co-infection of wheat by BMV and BSMV resulted in BMV-like symptoms with a drastic reduction in BSMV genomic RNA copies and coat protein accumulation, suggesting an antagonism-like effect exerted by BMV toward BSMV. However, co-infection of either BMV or BSMV with WSMV or TriMV led to more severe disease than singly infected wheat, but with a decrease or no significant change in titers of interacting viruses in the presence of BMV or BSMV, respectively. These results were in stark contrast with exacerbated disease phenotype accompanied with enhanced virus titers caused by WSMV and TriMV co-infection. Co-infection of wheat by WSMV, TriMV, and BMV or BSMV resulted in enhanced synergistic disease accompanied by increased accumulation of TriMV and BMV but not WSMV or BSMV. Quadripartite interactions in co-infected wheat by all four viruses resulted in very severe disease synergism, leading to the death of the most infected plants, but paradoxically, a drastic reduction in BSMV titer. Our results indicate that interactions among different viruses infecting the same plant host are more complex than previously thought, do not always entail increases in virus titers, and likely involve multiple mechanisms. These findings lay the foundation for additional mechanistic dissections of synergistic interactions among unrelated plant viruses.

Zika virus-like particle vaccine protects AG129 mice and rhesus macaques against Zika virus.

BACKGROUND: Zika virus (ZIKV), a mosquito-borne flavivirus, is a re-emerging virus that constitutes a public health threat due to its recent global spread, recurrent outbreaks, and infections that are associated with neurological abnormalities in developing fetuses and Guillain-Barré syndrome in adults. To date, there are no approved vaccines against ZIKV infection. Various preclinical and clinical development programs are currently ongoing in an effort to bring forward a vaccine for ZIKV. METHODOLOGY/PRINCIPLE FINDINGS: We have developed a ZIKV vaccine candidate based on Virus-Like-Particles (VLPs) produced in HEK293 mammalian cells using the prM (a precursor to M protein) and envelope (E) structural protein genes from ZIKV. Transient transfection of cells via plasmid and electroporation produced VLPs which were subsequently purified by column chromatography yielding approximately 2mg/L. Initially, immunogenicity and efficacy were evaluated in AG129 mice using a dose titration of VLP with and without Alhydrogel 2% (alum) adjuvant. We found that VLP with and without alum elicited ZIKV-specific serum neutralizing antibodies (nAbs) and that titers correlated with protection. A follow-up immunogenicity and efficacy study in rhesus macaques was performed using VLP formulated with alum. Multiple neutralization assay methods were performed on immune sera including a plaque reduction neutralization test, a microneutralization assay, and a Zika virus Renilla luciferase neutralization assay. All of these assays indicate that following immunization, VLP induces high titer nAbs which correlate with protection against ZIKV challenge. CONCLUSIONS/SIGNIFICANCE: These studies confirm that ZIKV VLPs could be efficiently generated and purified. Upon VLP immunization, in both mice and NHPs, nAb was induced that correlate with protection against ZIKV challenge. These studies support translational efforts in developing a ZIKV VLP vaccine for evaluation in human clinical trials.

A replication-competent adenovirus-vectored influenza vaccine induces durable systemic and mucosal immunity.

BACKGROUNDTo understand the features of a replicating vaccine that might drive potent and durable immune responses to transgene-encoded antigens, we tested a replication-competent adenovirus type 4 encoding influenza virus H5 HA (Ad4-H5-Vtn) administered as an oral capsule or via tonsillar swab or nasal spray.METHODSViral shedding from the nose, mouth, and rectum was measured by PCR and culturing. H5-specific IgG and IgA antibodies were measured by bead array binding assays. Serum antibodies were measured by a pseudovirus entry inhibition, microneutralization, and HA inhibition assays.RESULTSAd4-H5-Vtn DNA was shed from most upper respiratory tract-immunized (URT-immunized) volunteers for 2 to 4 weeks, but cultured from only 60% of participants, with a median duration of 1 day. Ad4-H5-Vtn vaccination induced increases in H5-specific CD4+ and CD8+ T cells in the peripheral blood as well as increases in IgG and IgA in nasal, cervical, and rectal secretions. URT immunizations induced high levels of serum neutralizing antibodies (NAbs) against H5 that remained stable out to week 26. The duration of viral shedding correlated with the magnitude of the NAb response at week 26. Adverse events (AEs) were mild, and peak NAb titers were associated with overall AE frequency and duration. Serum NAb titers could be boosted to very high levels 2 to 5 years after Ad4-H5-Vtn vaccination with recombinant H5 or inactivated split H5N1 vaccine.CONCLUSIONReplicating Ad4 delivered to the URT caused prolonged exposure to antigen, drove durable systemic and mucosal immunity, and proved to be a promising platform for the induction of immunity against viral surface glycoprotein targets.TRIAL REGISTRATIONClinicalTrials.gov NCT01443936 and NCT01806909.FUNDINGIntramural and Extramural Research Programs of the NIAID, NIH (U19 AI109946) and the Centers of Excellence for Influenza Research and Surveillance (CEIRS), NIAID, NIH (contract HHSN272201400008C).

Influence of rest interval lengths on hypotensive response after strength training sessions performed by older men.

The purpose of this study was to compare the postexercise hypotensive response after different rest intervals between sets (1 and 2 minutes) in normotense older men. Seventeen older men (67.6 ± 2.2 years) with at least 1 year of strength training experience participated. After determination of 10 repetition maximum (10RM) loads for exercises, subjects performed 2 different strength training sessions. On the first day, volunteers performed 3 sets of 10 repetitions per exercise at 70% 10RM, with 1 or 2 minutes' rest interval between sets depending on random assignment. On the second day, the procedures were similar but with the other rest interval. There was no difference in systolic and diastolic blood pressure between rest intervals at any time point measure. Before 1- and 2-minute sessions, the systolic blood pressure values were 122.7 ± 6.0 and 123.2 ± 3.7 mm Hg, and diastolic blood pressure values were 80.5 ± 5.6 and 82.0 ± 3.7 mm Hg, respectively. Both 1 and 2 minute sessions still presented reduced values for systolic blood pressure after 60 minutes (102.9 ± 6.9 and 106.7 ± 5.4 mm Hg, respectively), while the diastolic blood pressure presented significant reductions for 50 minutes after a 1 minute session (12.1 to 5.6 mm Hg) and for 60 minutes after the 2 minute session (13.3 to 6.5 mm Hg). Additionally, the systolic and diastolic blood pressure effect size data demonstrated higher magnitudes at all time point measures after the 2-minute rest sessions. These results suggest a poststrength training hypotensive response for both training sessions in normotense older men, with higher magnitudes for the 2-minute rest session. Our findings suggest a potentially positive health benefit of strength training.

Modified Adenovirus Prime-Protein Boost Clade C HIV Vaccine Strategy Results in Reduced Viral DNA in Blood and Tissues Following Tier 2 SHIV Challenge.

Designing immunogens and improving delivery methods eliciting protective immunity is a paramount goal of HIV vaccine development. A comparative vaccine challenge study was performed in rhesus macaques using clade C HIV Envelope (Env) and SIV Gag antigens. One group was vaccinated using co-immunization with DNA Gag and Env expression plasmids cloned from a single timepoint and trimeric Env gp140 glycoprotein from one of these clones (DNA+Protein). The other group was a prime-boost regimen composed of two replicating simian (SAd7) adenovirus-vectored vaccines expressing Gag and one Env clone from the same timepoint as the DNA+Protein group paired with the same Env gp140 trimer (SAd7+Protein). The env genes were isolated from a single pre-peak neutralization timepoint approximately 1 year post infection in CAP257, an individual with a high degree of neutralization breadth. Both DNA+Protein and SAd7+Protein vaccine strategies elicited significant Env-specific T cell responses, lesser Gag-specific responses, and moderate frequencies of Env-specific TFH cells. Both vaccine modalities readily elicited systemic and mucosal Env-specific IgG but not IgA. There was a higher frequency and magnitude of ADCC activity in the SAd7+Protein than the DNA+Protein arm. All macaques developed moderate Tier 1 heterologous neutralizing antibodies, while neutralization of Tier 1B or Tier 2 viruses was sporadic and found primarily in macaques in the SAd7+Protein group. Neither vaccine approach provided significant protection from viral acquisition against repeated titered mucosal challenges with a heterologous Tier 2 clade C SHIV. However, lymphoid and gut tissues collected at necropsy showed that animals in both vaccine groups each had significantly lower copies of viral DNA in individual tissues compared to levels in controls. In the SAd7+Protein-vaccinated macaques, total and peak PBMC viral DNA were significantly lower compared with controls. Taken together, this heterologous Tier 2 SHIV challenge study shows that combination vaccination with SAd7+Protein was superior to combination DNA+Protein in reducing viral seeding in tissues in the absence of protection from infection, thus emphasizing the priming role of replication-competent SAd7 vector. Despite the absence of correlates of protection, because antibody responses were significantly higher in this vaccine group, we hypothesize that vaccine-elicited antibodies contribute to limiting tissue viral seeding.

Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans.

Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4(+) and CD8(+) T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.