Comparison of Levels of Nasal, Salivary, and Plasma Antibody to Severe Acute Respiratory Syndrome Coronavirus 2 During Natural Infection and After Vaccination.

BACKGROUND: Most studies of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) measure antibody or cellular responses in blood; however, the virus infects mucosal surfaces in the nose and conjunctivae and infectious virus is rarely if ever present in the blood. METHODS: We used luciferase immunoprecipitation assays to measure SARS-CoV-2 antibody levels in the plasma, nose, and saliva of infected persons and vaccine recipients. These assays measure antibody that can precipitate the SAR-CoV-2 spike and nucleocapsid proteins. RESULTS: Levels of plasma anti-spike antibody declined less rapidly than levels of anti-nucleocapsid antibody in infected persons. SARS-CoV-2 anti-spike antibody levels in the nose declined more rapidly than antibody levels in the blood after vaccination of infected persons. Vaccination of previously infected persons boosted anti-spike antibody in plasma more than in the nose or saliva. Nasal and saliva anti-spike antibody levels were significantly correlated with plasma antibody in infected persons who had not been vaccinated and after vaccination of uninfected persons. CONCLUSIONS: Persistently elevated SARS-CoV-2 antibody in plasma may not indicate persistence of antibody at mucosal sites such as the nose. The strong correlation of SARS-CoV-2 antibody in the nose and saliva with that in the blood suggests that mucosal antibodies are derived primarily from transudation from the blood rather than local production. While SARS-CoV-2 vaccine given peripherally boosted mucosal immune responses in infected persons, the increase in antibody titers was higher in plasma than at mucosal sites. Taken together, these observations indicate the need for development of mucosal vaccines to induce potent immune responses at sites where SARS-CoV-2 infection occurs. CLINICAL TRIALS REGISTRATION: NCT01306084.

Serum and Cervicovaginal Fluid Antibody Profiling in Herpes Simplex Virus-Seronegative Recipients of the HSV529 Vaccine.

Previous herpes simplex virus type 2 (HSV-2) vaccines have not prevented genital herpes. Concerns have been raised about the choice of antigen, the type of antibody induced by the vaccine, and whether antibody is present in the genital tract where infection occurs. We reported results of a trial of an HSV-2 replication-defective vaccine, HSV529, that induced serum neutralizing antibody responses in 78% of HSV-1-/HSV-2- vaccine recipients. Here we show that HSV-1-/HSV-2- vaccine recipients developed antibodies to epitopes of several viral proteins; however, fewer antibody epitopes were detected in vaccine recipients compared with naturally infected persons. HSV529 induced antibodies that mediated HSV-2-specific natural killer (NK) cell activation. Depletion of glycoprotein D (gD)-binding antibody from sera reduced neutralizing titers by 62% and NK cell activation by 81%. HSV-2 gD antibody was detected in cervicovaginal fluid at about one-third the level of that in serum. A vaccine that induces potent serum antibodies transported to the genital tract might reduce HSV genital infection.

A Randomized, Double-Blinded, Placebo-Controlled, Phase 1 Study of a Replication-Defective Herpes Simplex Virus (HSV) Type 2 Vaccine, HSV529, in Adults With or Without HSV Infection.

BACKGROUND: Herpes simplex virus 2 (HSV2) causes genital herpes in >400 million persons worldwide. METHODS: We conducted a randomized, double-blinded, placebo-controlled trial of a replication-defective HSV2 vaccine, HSV529. Twenty adults were enrolled in each of 3 serogroups of individuals: those negative for both HSV1 and HSV2 (HSV1-/HSV2-), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2-). Sixty participants received vaccine or placebo at 0, 1, and 6 months. The primary end point was the frequency of solicited local and systemic reactions to vaccination. RESULTS: Eighty-nine percent of vaccinees experienced mild-to-moderate solicited injection site reactions, compared with 47% of placebo recipients (95% confidence interval [CI], 12.9%-67.6%; P = .006). Sixty-four percent of vaccinees experienced systemic reactions, compared with 53% of placebo recipients (95% CI, -17.9% to 40.2%; P = .44). Seventy-eight percent of HSV1-/HSV2- vaccine recipients had a ≥4-fold increase in neutralizing antibody titer after 3 doses of vaccine, whereas none of the participants in the other serogroups had such responses. HSV2-specific CD4+ T-cell responses were detected in 36%, 46%, and 27% of HSV1-/HSV2-, HSV1±/HSV2+, and HSV1+/HSV2- participants, respectively, 1 month after the third dose of vaccine, and CD8+ T-cell responses were detected in 14%, 8%, and 18% of participants, respectively. CONCLUSIONS: HSV529 vaccine was safe and elicited neutralizing antibody and modest CD4+ T-cell responses in HSV-seronegative vaccinees. CLINICAL TRIALS REGISTRATION: NCT01915212.

Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice.IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were able to specifically neutralize HSV-1 infection in vitro via HVEM. Furthermore, we showed for the first time that HVEM-specific HSV-1 neutralizing antibodies protect mice from HSV-1 eye disease, indicating the critical role of HVEM in HSV-1 ocular infection.

Two-Parameter Elliptical Fitting Method for Short-Cavity Fiber Fabry⁻Perot Sensor Interrogation.

To solve the cavity interrogation problem of short cavity fiber Fabry⁻Perot sensors in white light spectral interrogation with amplified spontaneous emissions (ASEs) as the white light sources, a data processing method, using an improved elliptical fitting equation with only two undetermined coefficients, is proposed. Based on the method, the cavity length of a fiber Fabry⁻Perot sensor without a complete reflection spectrum period in the frequency domain can be interrogated with relatively high resolution. Extrinsic fiber Fabry⁻Perot air-gap sensors with cavity lengths less than 30 µm are used to experimentally verify the method, and are successfully interrogated with an accuracy better than 0.55%.

A Herpes Simplex Virus 2 (HSV-2) gD Mutant Impaired for Neural Tropism Is Superior to an HSV-2 gD Subunit Vaccine To Protect Animals from Challenge with HSV-2.

UNLABELLED: A recent phase 3 trial with soluble herpes simplex virus 2 (HSV-2) glycoprotein D (gD2t) in adjuvant failed to show protection against genital herpes. We postulated that live attenuated HSV-2 would provide more HSV antigens for induction of virus-specific antibodies and cellular immunity than would gD2t. We previously reported an HSV-2 mutant, HSV2-gD27, in which the nectin-1 binding domain of gD2 is altered so that the virus is impaired for infecting neural cells, but not epithelial cells, in vitro and is impaired for infecting dorsal root ganglia in mice (K. Wang, J. D. Kappel, C. Canders, W. F. Davila, D. Sayre, M. Chavez, L. Pesnicak, and J. I. Cohen, J Virol 86:12891-12902, 2012, doi:10.1128/JVI.01055-12). Here we report that the mutations in HSV2-gD27 were stable when the virus was passaged in cell culture and during acute infection of mice. HSV2-gD27 was attenuated in mice when it was inoculated onto the cornea, intramuscularly (i.m.), intravaginally, and intracranially. Vaccination of mice i.m. with HSV2-gD27 provided better inhibition of challenge virus replication in the vagina than when the virus was used to vaccinate mice intranasally or subcutaneously. Comparison of i.m. vaccinations with HSV2-gD27 versus gD2t in adjuvant showed that HSV2-gD27 induced larger reductions of challenge virus replication in the vagina and reduced latent viral loads in dorsal root ganglia but induced lower serum neutralizing antibody titers than those obtained with gD2t in adjuvant. Taken together, our data indicate that a live attenuated HSV-2 vaccine impaired for infection of neurons provides better protection from vaginal challenge with HSV-2 than that obtained with a subunit vaccine, despite inducing lower titers of HSV-2 neutralizing antibodies in the serum. IMPORTANCE: Genital herpes simplex is one of the most prevalent sexually transmitted diseases. Though HSV-2 disease is usually mild, it can be life threatening in neonates and immunocompromised persons. In addition, genital herpes increases the frequency of HIV infection and transmission. HSV-2 maintains a latent infection in sensory neurons and cannot be cleared with antiviral drugs. The virus frequently reactivates, resulting in virus shedding in the genital area, which serves as a source for transmission. A prophylactic vaccine is needed to prevent disease and to control the spread of the virus. Previous human trials of subunit vaccines have been unsuccessful. Here we report the results of vaccinating mice with a new type of live attenuated HSV-2 vaccine that is impaired for infection of neurons and provides better protection of mice than that obtained with a subunit vaccine. The strategy of altering the cell tropism of a virus is a new approach for a live attenuated vaccine.

Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

UNLABELLED: No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. IMPORTANCE: Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.

Genome Sequencing and Analysis of Geographically Diverse Clinical Isolates of Herpes Simplex Virus 2.

UNLABELLED: Herpes simplex virus 2 (HSV-2), the principal causative agent of recurrent genital herpes, is a highly prevalent viral infection worldwide. Limited information is available on the amount of genomic DNA variation between HSV-2 strains because only two genomes have been determined, the HG52 laboratory strain and the newly sequenced SD90e low-passage-number clinical isolate strain, each from a different geographical area. In this study, we report the nearly complete genome sequences of 34 HSV-2 low-passage-number and laboratory strains, 14 of which were collected in Uganda, 1 in South Africa, 11 in the United States, and 8 in Japan. Our analyses of these genomes demonstrated remarkable sequence conservation, regardless of geographic origin, with the maximum nucleotide divergence between strains being 0.4% across the genome. In contrast, prior studies indicated that HSV-1 genomes exhibit more sequence diversity, as well as geographical clustering. Additionally, unlike HSV-1, little viral recombination between HSV-2 strains could be substantiated. These results are interpreted in light of HSV-2 evolution, epidemiology, and pathogenesis. Finally, the newly generated sequences more closely resemble the low-passage-number SD90e than HG52, supporting the use of the former as the new reference genome of HSV-2. IMPORTANCE: Herpes simplex virus 2 (HSV-2) is a causative agent of genital and neonatal herpes. Therefore, knowledge of its DNA genome and genetic variability is central to preventing and treating genital herpes. However, only two full-length HSV-2 genomes have been reported. In this study, we sequenced 34 additional HSV-2 low-passage-number and laboratory viral genomes and initiated analysis of the genetic diversity of HSV-2 strains from around the world. The analysis of these genomes will facilitate research aimed at vaccine development, diagnosis, and the evaluation of clinical manifestations and transmission of HSV-2. This information will also contribute to our understanding of HSV evolution.

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

Painful pathways induced by TLR stimulation of dorsal root ganglion neurons.

We hypothesize that innate immune signals from infectious organisms and/or injured tissues may activate peripheral neuronal pain signals. In this study, we demonstrated that TLRs 3, 7, and 9 are expressed by human dorsal root ganglion neurons (DRGNs) and in cultures of primary mouse DRGNs. Stimulation of murine DRGNs with TLR ligands induced expression and production of proinflammatory chemokines and cytokines CCL5 (RANTES), CXCL10 (IP-10), IL-1α, IL-1ß, and PGE(2), which have previously been shown to augment pain. Further, TLR ligands upregulated the expression of a nociceptive receptor, transient receptor potential vanilloid type 1 (TRPV1), and enhanced calcium flux by TRPV1-expressing DRGNs. Using a tumor-induced temperature sensitivity model, we showed that in vivo administration of a TLR9 antagonist, known as a suppressive oligodeoxynucleotide, blocked tumor-induced temperature sensitivity. Taken together, these data indicate that stimulation of peripheral neurons by TLR ligands can induce nerve pain.

Detection of molluscum contagiosum virus (MCV) DNA in the plasma of an immunocompromised patient and possible reduction of MCV DNA with CMX-001.

Molluscum contagiosum virus (MCV) is a poxvirus that causes localized papules in healthy persons. We evaluated a woman with severe immunodeficiency and disseminated MCV. During treatment with CMX-001, an antiviral with activity against other poxviruses, MCV DNA was detected in 20% of plasma samples. When the patient was not receiving CMX-001, MCV DNA was detected in 50% of samples. We also noted improvement in warts on her fingers during CMX-001 therapy. Although MCV is caused by direct inoculation of virus into skin in healthy persons, in a severely immunocompromised person MCV DNA was present in blood and may spread by viremia.

Functional stability correlates with dynamic microbial networks in anammox process.

Understanding the relationship between dynamic microbial networks and functional stability is critical for the stable operation of anammox systems. Here, by operating an anammox reactor under constant condition over 250 days, it was found that the relative abundance of Planctomycetota gradually decreased while Chloroflexi and Proteobacteria increased, with stochasticity predominating the bacterial assembly as the reactor operation. Network analysis revealed a successional dynamic pattern of microbial interaction despite stable performance. The variation of subnetworks indicated Chloroflexi and Proteobacteria alternately played important role in anammox microbial network, and the negative relationship between anammox bacteria and heterotrophs could achieve a balance to keep functional stability under long-term operation. Furthermore, the identified keystone species mainly belonged to heterotrophs that were critical in maintaining network structure and system function. The results of this study revealed clear changing patterns of microbial community and network succession, which could provide valuable reference for other stably operated bioreactors.

Sex and prior exposure jointly shape innate immune responses to a live herpesvirus vaccine.

Background: Both sex and prior exposure to pathogens are known to influence responses to immune challenges, but their combined effects are not well established in humans, particularly in early innate responses critical for shaping subsequent outcomes. Methods: We employed systems immunology approaches to study responses to a replication-defective, herpes simplex virus (HSV) 2 vaccine in men and women either naive or previously exposed to HSV. Results: Blood transcriptomic and cell population profiling showed substantial changes on day 1 after vaccination, but the responses depended on sex and whether the vaccinee was naive or previously exposed to HSV. The magnitude of early transcriptional responses was greatest in HSV naive women where type I interferon (IFN) signatures were prominent and associated negatively with vaccine-induced neutralizing antibody titers, suggesting that a strong early antiviral response reduced the uptake of this replication-defective virus vaccine. While HSV seronegative vaccine recipients had upregulation of gene sets in type I IFN (IFN-α/ß) responses, HSV2 seropositive vaccine recipients tended to have responses focused more on type II IFN (IFN-γ) genes. Conclusions: These results together show that prior exposure and sex interact to shape early innate responses that then impact subsequent adaptive immune phenotypes. Funding: Intramural Research Program of the NIH, the National Institute of Allergy and Infectious Diseases, and other institutes supporting the Trans-NIH Center for Human Immunology, Autoimmunity, and Inflammation. The vaccine trial was supported through a clinical trial agreement between the National Institute of Allergy and Infectious Diseases and Sanofi Pasteur. Clinical trial number: NCT01915212.

Identification of viral microRNAs expressed in human sacral ganglia latently infected with herpes simplex virus 2.

Deep sequencing of small RNAs isolated from human sacral ganglia latently infected with herpes simplex virus 2 (HSV-2) was used to identify HSV-2 microRNAs (miRNAs) expressed during latent infection. This effort resulted in the identification of five distinct HSV-2 miRNA species, two of which, miR-H3/miR-I and miR-H4/miR-II, have been previously reported. Three novel HSV-2 miRNAs were also identified, and two of these, miR-H7 and miR-H9, are derived from the latency-associated transcript (LAT) and are located antisense to the viral transcript encoding transactivator ICP0. A third novel HSV-2 miRNA, miR-H10, is encoded within the unique long (U(L)) region of the genome, 3' to the U(L)15 open reading frame, and is presumably excised from a novel, latent HSV-2 transcript distinct from LAT.

An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor.

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.

VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease.

Mitochondrial stress releases mitochondrial DNA (mtDNA) into the cytosol, thereby triggering the type Ι interferon (IFN) response. Mitochondrial outer membrane permeabilization, which is required for mtDNA release, has been extensively studied in apoptotic cells, but little is known about its role in live cells. We found that oxidatively stressed mitochondria release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane. Furthermore, the positively charged residues in the N-terminal domain of VDAC1 interact with mtDNA, promoting VDAC1 oligomerization. The VDAC oligomerization inhibitor VBIT-4 decreases mtDNA release, IFN signaling, neutrophil extracellular traps, and disease severity in a mouse model of systemic lupus erythematosus. Thus, inhibiting VDAC oligomerization is a potential therapeutic approach for diseases associated with mtDNA release.

Glutamine supplementation suppresses herpes simplex virus reactivation.

Chronic viral infections are difficult to treat, and new approaches are needed, particularly those aimed at reducing reactivation by enhancing immune responses. Herpes simplex virus (HSV) establishes latency and reactivates frequently, and breakthrough reactivation can occur despite suppressive antiviral therapy. Virus-specific T cells are important to control HSV, and proliferation of activated T cells requires increased metabolism of glutamine. Here, we found that supplementation with oral glutamine reduced virus reactivation in latently HSV-1-infected mice and HSV-2-infected guinea pigs. Transcriptome analysis of trigeminal ganglia from latently HSV-1-infected, glutamine-treated WT mice showed upregulation of several IFN-γ-inducible genes. In contrast to WT mice, supplemental glutamine was ineffective in reducing the rate of HSV-1 reactivation in latently HSV-1-infected IFN-γ-KO mice. Mice treated with glutamine also had higher numbers of HSV-specific IFN-γ-producing CD8 T cells in latently infected ganglia. Thus, glutamine may enhance the IFN-γ-associated immune response and reduce the rate of reactivation of latent virus infection.

A novel HSV-2 subunit vaccine induces GLA-dependent CD4 and CD8 T cell responses and protective immunity in mice and guinea pigs.

BACKGROUND/OBJECTIVES: There is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection. METHODS: We developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination. RESULTS: Vaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score. CONCLUSIONS: Taken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.

Isolation, Selection, and Efficacy of Pochonia chlamydosporia for Control of Rotylenchulus reniformis on Cotton.

ABSTRACT The reniform nematode, Rotylenchulus reniformis, is a serious threat to cotton (Gossypium hirsutum) production in the United States, causing an annual loss of about $80 million. The objective of this study was to isolate fungi from eggs of R. reniformis and select potential biocontrol agents for R. reniformis on cotton. We focused on the fungus Pochonia chlamydosporia because it suppresses root-knot and cyst nematodes and because preliminary data indicated that it was present in Arkansas cotton fields. Soil samples were collected from six cotton fields in Jefferson County, Arkansas. A total of 117 isolates of the nematophagous fungus P. chlamydosporia were obtained. In an in vitro test, 105 of the 117 isolates parasitized fewer than 15% of R. reniformis eggs, but 12 isolates parasitized between 16 and 35% of the eggs. These 12 isolates produced from 6.8 x 10(4) to 6.9 x 10(5) chlamydospores per gram of medium in vitro, and chlamydospore production was similar on rice grain and corn grain media. In two greenhouse experiments, a single application of isolate 37 (5,000 chlamydospores per gram of soil) significantly reduced the numbers of R. reniformis on cotton roots and in soil. The three isolates (37, 26, and 14) that parasitized the most eggs in vitro were also the most effective in suppressing numbers of R. reniformis and in increasing cotton growth in the greenhouse.

Suppression of Rotylenchulus reniformis on Cotton by the Nematophagous Fungus ARF.

The reniform nematode, Rotylenchulus reniformis Linford &Oliveira, has become a serious threat to cotton (Gossypium hirsutum L.) production in the United States during the past decade. The objective of this study is to isolate fungi from eggs of R. reniformis and select potential biological control agents for R. reniformis on cotton. Soil samples were collected from cotton fields located in Jefferson County, Arkansas. Eight genera of fungi were included in the 128 fungal isolates obtained, and among them were five strains of the nematophagous fungus ARF. The mtDNA RFLP pattern, colony growth characteristics, and pathogenicity indicate the five ARF isolates represent one described strain and one new strain. Light and electron microscopic observations suggest ARF is an active parasite of R. reniformis, with parasitism ranging from 48% to 79% in in vitro tests. Three greenhouse experiments demonstrated ARF successfully suppressed the number of reniform nematodes during the first and second generation of the nematode. Reductions in numbers of R. reniformis on the roots for the seven application rates of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ARF were 87%, 92%, 94%, 96%, 97%, 98%, and and 98%, respectively.